Decomposition rate of extraradical hyphae of arbuscular mycorrhizal fungi decreases rapidly over time and varies by hyphal diameter and season

Soil Biology and Biochemistry 136 (2019) 107533

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Decomposition rate of extraradical hyphae of arbuscular mycorrhizal fungi decreases rapidly over time and varies by hyphal diameter and season

T

Holger Schäfera,*, Masako Dannouraa,b, Mioko Atakac, Akira Osawaa,b a

Laboratory of Ecosystem Production and Dynamics, Graduate School of Global Environmental Studies, Kyoto University, Kyoto, 606-8502, Japan Laboratory of Forest Utilization, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan c Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto, 611-0011, Japan b

ARTICLE INFO

ABSTRACT

Keywords: Forest carbon cycle Arbuscular mycorrhiza Mycorrhizal fungal hyphae In-growth mesh bags Litter decomposition Hyphal length density

Understanding how extraradical mycorrhizal fungal hyphae (EMH) regulate the cycling and retention of plantassimilated carbon (C) in forest soils requires estimation of the production, mortality, and decomposition of EMH. To do this, the use of mass-balance models in combination with hyphal in-growth mesh bags (“in-growth bags”) was proposed in recent studies. However, poor knowledge on the decomposition of field-grown EMH prevents confirmation of assumed EMH decomposition dynamics. In this study, we determined the decrease in hyphal length density of field-grown EMH of arbuscular mycorrhizal fungi (AM) over eight months in in-growth bags incubated in a warm-temperate Chamaecyparis obtusa forest, to study EMH decomposition under field conditions. We used exponential decay models to describe the changes in the decomposition rate of EMH over the incubation time, between EMH diameter classes, and between seasons. A rapid decrease of the decomposition rate of EMH within two months from 2.5 to 0.1 month−1 was estimated, corresponding to an increase in half-life from 0.3 to 7 months. Furthermore, significant differences in the initial maximum decomposition rate were estimated between fine (1.6 month−1; minimum half-life: 0.4 months) and coarse EMH (3.1 month−1; minimum half-life: 0.2 months) and between incubations during spring-summer (April and August; 2.7 month−1; minimum half-life: 0.3 months) and autumn-winter (October and February; 1.1 month−1; minimum half-life: 0.6 months). This large variability in the decomposition rates of EMH of AM fungi has to be considered in mass-balance models to estimate C fluxes between plants, soil, and the atmosphere.

1. Introduction Mycorrhizal fungi are ubiquitous plant symbionts (Brundrett, 2009; Smith and Read, 2008). In exchange for benefits in plant nutrient and water acquisition, up to 22% of the total plant-assimilated carbon (C) has been estimated to be allocated to mycorrhizal fungi in forests (Hobbie, 2006). To extract nutrients and water from soils, mycorrhizal fungi produce extraradical mycorrhizal fungal hyphae (EMH). In boreal and temperate forests, the production of EMH alone accounts for 2.5–14% of the net primary production (Allen and Kitajima, 2014; Ekblad et al., 2016, 2013; Hobbie, 2006), making EMH major contributors to the plant – soil C flux. Studies at more than 140 sites have quantified EMH production in temperate and boreal forests of ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) tree species (Allen and Kitajima, 2014; Bakker et al., 2015; Ekblad et al., 2016, 2013; Hagenbo et al., 2017; Hendricks et al., 2016; Schäfer et al., 2018). Understanding how EMH regulate the cycling and retention of

*

plant-assimilated C in forest soils, however, requires field observations of the decomposition of EMH. Previous studies have shown that the turnover of EMH may vary between one and ten times per year in boreal and temperate forests (Ekblad et al., 2016; Hagenbo et al., 2017; Hendricks et al., 2016), indicating large differences in EMH mortality and decomposition among sites and years. To estimate EMH production, mortality, and decomposition recent studies proposed the use of mass-balance models on data of incubation studies with sand-filled hyphal in-growth mesh bags (abbreviated as “in-growth bags” hereafter; Ekblad et al., 2016; Hagenbo et al., 2017; Li and King, 2018). Ingrowth bags are a commonly used tool to estimate the production of EMH (Ekblad et al., 2013). They are made from a fine mesh that enables in-growth of hyphae but not roots (25–50 μm mesh size) and are filled with sand which effectively limits the colonization by non-mycorrhizal fungal hyphae (Wallander et al., 2013, 2001). After incubation in soil for 2–12 months, in-growth bags are collected, and the EMH production is estimated from the amount of hyphae recovered at the time of

Corresponding author. E-mail address: [email protected] (H. Schäfer).

https://doi.org/10.1016/j.soilbio.2019.107533 Received 8 March 2019; Received in revised form 5 July 2019; Accepted 8 July 2019 Available online 10 July 2019 0038-0717/ © 2019 Elsevier Ltd. All rights reserved.

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collection compared to that at the start of incubation. Additional estimates of EMH decomposition with in-growth bags provide an indication of the EMH turnover. In the first study that estimated EMH decomposition with in-growth bags, Ekblad et al. (2016) observed that a large proportion of EMH was dead at collection of in-growth bags incubated for 4.5–29 months in a warm-temperate Pinus taeda stand. An exponential decay of dead EMH with a constant decomposition rate k (month−1) was assumed during in-growth bag incubation (Jenny et al., 1949; Olson, 1963):

EMHt = EMH0 e kt with k = ln(EMHt / EMH0 )/t

To predict the temporal change in EMH decomposition, Li and King (2018) proposed a more complex mass-balance model which assumes cohorts of decomposing EMH, due to different time points of EMH death, and an exponential decrease of the decomposition rate of each EMH cohort. A decomposition model with an exponentially decreasing decomposition rate was found to describe the decomposition of several litter types (Carpenter, 1982; Godshalk and Wetzel, 1978; Rovira and Rovira, 2010). EMH decomposition dynamics are, however, only known from a three-month study with laboratory-grown EMH incubated in forest soil (Fernandez and Koide, 2014). As field observations of the decomposition of field-grown EMH are lacking, it is unclear whether the previously estimated decomposition dynamics actually apply and whether there is seasonal variation and variation due to hyphal diameter. In this study, we followed the decomposition of field-grown EMH of arbuscular mycorrhizal fungi over eight months in in-growth bags incubated in the soil of a warm-temperate Chamaecyparis obtusa (hinoki cypress, evergreen conifer) stand in Central Japan. For this, the hyphal length densities (HLDs) of EMH litter in small litter bags contained in in-growth bags were measured before and after incubation for 0.5–8 months. We tested (1) whether the amount of EMH litter after two months was significantly different between litter bags with and without a surrounding in-growth bag, (2) whether EMH decomposition is better described by a decomposition model with a constant or an exponentially decreasing decomposition rate, and (3) how the decomposition rate varies between seasons and fine and coarse EMH litter.

(1)

where EMHt is the fraction (%) remaining at time t (months) of the initial amount EMH0 (100%). The decomposition rate k, thereby, relates the instantaneous change of EMHt at time t (% month−1) to EMHt (%) in the linear first-order differential equation dEMHt /dt = kEMHt (Rovira and Rovira, 2010). A lower decomposition rate indicates a slower decomposition. Estimated decomposition rates of 0.11–0.13 month−1 (control plots; Ekblad et al., 2016) indicated a half-life time of 5.4–6.6 months and an EMH decomposition loss of 12% or less within one month. The decomposition loss was low compared to the one reported for laboratory-grown EMH litter placed in forest soil of 20–60% dry mass within one month right after placement (Fernandez and Koide, 2014, 2012). It was suggested that EMH decomposition in in-growth bags may have been slow due to low abundance of saprotrophs in sandfilled in-growth bags or redistribution of nitrogen to other EMH before death of EMH and hence slow decomposition due to N-limitation (Ekblad et al., 2016). However, limited field data on EMH decomposition of ECM fungi and particularly AM fungi have since prevented confirmation of these suggestions and limit the reliability of mass-balance modeling results. The decomposition of many types of litter, including litter of mycorrhizal fungal fruit bodies and EMH, was found to slow down over time (Brabcová et al., 2016; Fernandez and Koide, 2014; Li and King, 2018; Rovira and Rovira, 2010). One may, therefore, also consider the timing of observation, when assessing EMH decomposition. For laboratory-grown EMH litter placed in soil (Fernandez and Koide, 2014, 2012), large decomposition losses were estimated at the initial stage of decomposition of EMH which had still been growing in the laboratory. For field-grown EMH in in-growth bags (Ekblad et al., 2016), however, EMH grew into bags, died, and started to decompose anytime during incubation for 4.5–29 months. Hence, many dead EMH may have been in later stages of decomposition at times of in-growth bag collection, resulting in the low estimates of EMH decomposition rates. For improved estimation of EMH decomposition in mass-balance models, initial as well as later stages of EMH decomposition may need to be measured in the field and described. Slow decomposition at later stages is commonly explained by a change in litter properties as decomposition proceeds (Minderman, 1968; Rovira and Rovira, 2010; Wieder and Lang, 1982). Labile compounds (i.e. sugars, glycogen, starch) decompose more rapidly than recalcitrant ones (i.e. cellulose, lignin); as the former decrease in their amount relative to the latter, litter decomposition slows. Nevertheless, labile and recalcitrant compounds may release or evolve into compounds of a different labile and recalcitrant quality during decomposition (Rovira and Rovira, 2010). In addition, labile and recalcitrant compounds are unlikely to decompose independently from each other (Rovira and Rovira, 2010). In case of laboratory-grown EMH litter, even small variation in the concentration of melanin and chitin significantly affect decomposition (Fernandez and Koide, 2014, 2012). Furthermore, EMH decomposition may depend on the thickness of EMH (Langley and Hungate, 2003; Steinberg and Rillig, 2003). EMH decomposition dynamics may thus not only be predictable based on their chemical composition but also other factors such as compound interactions or diameter.

2. Materials and methods 2.1. Site description The study was conducted in a 340 m2 plot within the Chamaecyparis obtusa Endl. (Pinales, Cupressaceae) plantation forest of the Kiryu Experimental Watershed in Otsu City, Shiga Prefecture, Japan (34°58′ N, 136°00’ E) planted in 1959. The understory is dominated by evergreen broad-leaf shrubs, mainly Eurya japonica Thunb. (Ericales, Pentaphylacaceae). Association of C. obtusa and E. japonica with arbuscular mycorrhizal (AM) fungi had previously been demonstrated by assessment of fungal morphology (Maeda, 1954; Yamato and Iwasaki, 2002) and community composition and structure analysis based on DNA sequencing (Toju et al., 2014; Yamato et al., 2011). The AM symbiosis is common in the family Cupressaceae and, with exception of Ericacea and Diapensiaceae, in the families of the order Ericales (Wang and Qiu, 2006). Roots of C. obtusa were reported to be primarily colonized by AM fungi of the genus Glomus (Yamato et al., 2011). The mean air temperature and mean annual precipitation at the study site were 13.5 °C and 1674 mm, respectively, for the period 2001 to 2016 (Epron et al., 2018). The soil at the study site is a cambisol (IUSS Working Group WRB, 2015; Obara et al., 2015) on granite bed rock (Kosugi et al., 2013; Sakabe et al., 2015). During the study period from April 2017 to April 2018, daily mean soil temperature as measured at 5 cm soil depth (RT-1, Decagon Devices, Pullman, USA) showed distinct seasonal variation with a maximum in August and a minimum in February (Fig. 1). The daily mean soil water content at 5 cm soil depth did not vary between spring-summer and autumnwinter (EC-5, Decagon Devices, Pullman, USA; Fig. 1). 2.2. Preparation of in-growth bags containing litter and control bags To determine EMH decomposition in in-growth bags, we added small litter and control bags (Fig. 2). Field-grown EMH litter was assembled from in-growth bags incubated by Schäfer et al. (2018) for two months into the soil of a nearby C. obtusa plantation forest planted in the 1930s with a similar tree species composition as the plot used for this incubation study. The sand containing the EMH litter was kept frozen at −20 °C until litter bag assembly. At the site of EMH litter 2

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small fraction of EMH of ECM fungi. Nevertheless, hyphae with typical morphological features of EMH of AM fungi such as irregular or no septation and uneven diameters (Friese and Allen, 1991) were abundantly found by Schäfer et al. (2018) in the in-growth bags whose contents were used for the present study. In-growth of non-mycorrhizal fungal hyphae into in-growth bags is unavoidable under field conditions (Wallander et al., 2013, 2001). Schäfer et al. (2018) calculated the contribution of non-mycorrhizal fungal hyphae to the hyphae in in-growth bags as the fraction of the hyphal length (HL) production in control bags, incubated in root-trenched areas where EMH were deprived of plant C input, relative to the HL production in in-growth bags. After excluding in-growth bags from locations and collection dates with high contributions by non-mycorrhizal fungal hyphae, we estimated a total hyphal length of ca. 700 m to be available for litter bag preparation (ca. 6 m per bag for a total of 110–120 litter bags). The contribution of non-mycorrhizal fungal hyphae to the total available hyphal length was estimated at 18%, based on Schäfer et al. (2018). Keeping this contribution of non-mycorrhizal fungi in mind, the litter is, for convenience, referred to as EMH litter hereafter. The EMH litter was mixed with 500 ml of distilled water and homogenized with 8000 rotations per minute for 1 min (AHG-160, As One, Osaka, Japan) with a shaft generator (HT-1018) to untangle hyphal aggregates. While mixing on a magnet stirrer, 480 portions of the hyphal suspension, each 1 ml in volume, were equally distributed in 240 capped microtubes (2 ml volume, GDMST-2ML, As One, Osaka, Japan) using a glass pipette (2 ml volume). Damage from homogenization and freezing could not be avoided and may have accelerated initial EMH decomposition. After homogenization by blending, hyphal fragments retained a length of ca. 0.2–1.3 mm as determined at 50 × magnification using a dissecting microscope (SZX12, Olympus, Tokyo, Japan). Cylinder-shaped in-growth bags (10 cm length × 2.8 cm diameter) and cylinder-shaped litter and control bags (5 cm length × 1 cm diameter) were made from nylon mesh cuttings with mesh sizes of 50 and 1 μm, respectively, using a hot glue pistol. The 1 μm mesh size of litter and control bags was chosen to reduce in-growth of new hyphae and to avoid washing out of fragmented EMH litter. EMH litter decomposition was expected to depend more on bacterial than on fungal degradation, in line with observations for mycorrhizal fruit bodies (Brabcová et al., 2016). Collembola, mites, nematodes and, other soil organisms with body sizes > 1 μm may feed on EMH of AM fungi, or damage them (Gange and Brown, 2002; Hodge, 2000). Preventing such organisms from accessing the EMH litter may, therefore, have reduced the decomposition rate of the EMH litter in litter bags compared to natural soil. Nevertheless, collembola and mites, for instance, show feeding preferences for other types of fungi over AM fungi (Gange and Brown, 2002; Tiunov and Scheu, 2005), suggesting limited effect of their absence on the inferred decomposition rates of EMH of AM fungi. Litter bags were filled with 3 g of dry granite sand (grain size of 0.16–2.0 mm, C content not detectable by CN analysis, Daiki, Japan). For hyphal addition, the contents of soil suspensions of two microtubes were poured on the sand, and another 3 g of dry granite sand were added on top. After closing the top seal of each litter bag, a control bag of the same design was attached to its back seal with hot glue to correct for hyphal in-growth. In between of the litter and control bag a narrow plastic strip was included and later connected to the top seal of the ingrowth bag to keep the bags positioned at a central height (Fig. 2). The space of the in-growth bags that remained empty, after adding the pair of litter and control bags, was filled with 83 g of dry granite sand.

Fig. 1. Seasonal variation of the daily mean soil temperature in the forest (black line; measured 5 cm below the surface) and of the daily mean soil water content (grey line; measured 5 cm below the surface) during the study period from April 5, 2017 to April 5, 2018. Grey horizontal lines at the top represent incubation periods of in-growth bags containing litter of extraradical mycorrhizal fungal hyphae (EMH).

Fig. 2. Design of the hyphal in-growth mesh bag (‘in-growth bag’) containing bags with (‘litter bag’) and without (‘control bag’) extraradical mycorrhizal fungal hyphae (EMH). The thick dark line represents a narrow plastic strip by which the litter and control bags are hung to the top seal of the in-growth bag to hold them in position in the center.

2.3. Incubation of in-growth bags

production, 87% of the annual stem wood production was contributed by AM tree species (Schäfer et al., 2018). There are, however, also several ectomycorrhizal (ECM) oak trees of the species Quercus serrata Murray. The employed EMH litter may, therefore, have also contained a

To determine the initial amount of field-grown EMH litter in the litter bags, six litter bags were directly subjected to hyphal extraction after they had been constructed. To assess the decomposition of the 3

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EMH litter, 36 in-growth bags containing a litter and control bag were incubated in the field from April 2017 for 0.5, 1, 1.5, 2, 4, and 8 months. Six in-growth bags were collected at each sampling time (Fig. 1). To assess the effect of the surrounding in-growth bag on EMH litter decomposition, four additional in-growth bags and ten pairs of litter and control bags without surrounding in-growth bags were incubated in the field from April 2017 and collected two months later. To assess the decomposition of the EMH litter in other seasons, 18 ingrowth bags were incubated from August 2017, October 2017, and February 2018, respectively, for 0.5, 1, and 2 months. Six in-growth bags were collected at each sampling time (Fig. 1). For in-growth bag incubation, holes of 10 cm depth and 2.8 cm diameter were drilled into the organo-mineral layer, using a stainlesssteel pipe with a sharpened edge, at random locations along ten parallel, 15 m-long transects. For the incubation of the pairs of litter and control bags without surrounding in-growth bags, smaller holes of 7.5 cm depth and 2 cm diameter were prepared and filled up with part of the removed soil after bag insertion. Bags were collected by carefully pulling them out of the soil and put into grip seal plastic bags for transportation in an ice box to the laboratory, where they were stored frozen at −20 °C until further analysis. Three in-growth bags were lost because of damage by wildlife. In the laboratory, in-growth bags were opened at the top seal. The litter and control bags were instantly put in a freezer (−20 °C) and stored until further analysis. The granite sand of the in-growth bags was transferred to a clean grip seal plastic bag for homogenization by careful mixing. A subsample of ca. 10 g was then taken for water content estimation (at 65 °C for 48 h), before freezing again until further analysis.

2.5. Hyphal length measurement The amount of extracted hyphae was quantified in terms of hyphal length density (HLD), a common indicator of the amount of EMH in ingrowth bags (Bakker et al., 2015; Wallander et al., 2013, 2004). EMH were assumed to have decomposed, when they were not detected by hyphal extraction and length measurement. Hence, reported amounts of decomposed EMH may differ from the actually mineralized ones. The C content of small amounts of extracted hyphae could not be accurately measured with the available equipment. Each hyphal suspension was carefully filtered through a glass filter (GA-100, 25 mm, Advantec MFS, Tokyo, Japan) using a filter holder (Swinnex 37, Merck Millipore, Darmstadt, Germany) attached to a 10 ml syringe (SS-10LZ, Terumo, Tokyo, Japan). Glass filters were placed under a dissecting microscope (Olympus SZX12, Olympus, Tokyo, Japan) with a camera attached (Canon EOS 500D, Canon, Tokyo, Japan). Using 50 × magnification, 20 digital images (1.6 × 2.4 mm photographed area) were taken, which accounted for ca. 20% of each filter's total area. Detection of finer hyphae on the images was confirmed by assessment at magnifications of up to 90 × . The hyphal length (mm) on each filter was estimated by counting the hyphal intersections with a 0.4 × 0.4 mm grid imposed on the images, following Tennant (1975). The programming language R (R Core Team, 2017) with the package jpeg (Urbanek, 2014) was used to draw grid lines and the package zoom (Barbu, 2013) to zoom along grid lines. HLDs of EMH litter (mm g−1 dry sand) were calculated by subtracting the HLDs in the control bags from those in the corresponding litter bags. Based on the relationship between the hyphal loss during hyphal extraction (% of HLD of EMH litter) and the HLD of EMH litter in 16 litter bags, hyphal loss during hyphal extraction was computed for all 110 study units and added to the HLDs of the EMH litter. A hyphal loss of 5% during hyphal extraction was assumed for the control as well as ingrowth bags.

2.4. Hyphal extraction Hyphae were extracted from sand of the surrounding in-growth bag as well as the litter and control bags by suspending the sand in water as described in Schäfer et al. (2018). Hyphae float near the water surface, when the sand-water suspension is stirred, enabling separation from sand grains which quickly sediment (Bakker et al., 2015; Ekblad et al., 2016; Wallander et al., 2004). Briefly, each portion of sand (6 g from litter and control bags and 20 g from the surrounding in-growth bags) was mixed with water in a 50 ml glass vial. The sand in the litter and control bags was directly washed out into the glass vial with a squeeze bottle after opening the top seal. Removal of all hyphae from the mesh was confirmed by observation at 50 × magnification after emptying the bags. After stirring the suspension, the floating hyphae were carefully poured onto a test sieve (53 μm mesh size, 20 cm frame diameter, 7 cm frame height). This procedure was repeated until the water remained mostly clear from fine particles after stirring. No hyphae were observed among the remaining coarse sand grains following this procedure. The captured organic material containing the hyphae was then washed to the edge between the sieve mesh and frame. Water was added to the assembled material several times, and the resulting suspension with floating hyphae was transferred to a clean sample tube each time. Hyphae were stained with methylene blue, poured again on the test sieve, and transferred to 20 ml of clear water in a 20 ml plastic bottle. The described hyphal extraction procedure led to loss of 5% of the hyphae through the test sieve (Schäfer et al., 2018). However, as hyphal loss during hyphal extraction may vary with the decomposition state of EMH, we estimated it once more for 16 litter bags incubated for either 0.5, 1, or 2 months. To collect lost hyphae, all water used for hyphal extraction from each litter bag was collected below the test sieve in a tray and then carefully filtered through an open, rectangular mesh pocket (1 μm mesh size) over a glass funnel. Each mesh pocket was sealed with a plastic bag sealer, stained with methylene blue in a glass vial, washed carefully with clear water, and cut open. The filtrate with the stained hyphal fragments was transferred to 20 ml of clear water in a plastic sample bottle (Schäfer et al., 2018).

2.6. Separation into fine and coarse EMH litter Hyphal diameters were measured on 10 of the 20 images taken of each of the six litter bags that were not incubated and all 50 pairs of litter and control bags incubated from April. Using the image processing software ImageJ (version 1.51k; Schneider et al., 2012), a cross of two intersecting lines of 1.6 and 2.4 mm length was drawn through the center of each image and the thickness of all touching and intersecting hyphae were measured. A circular cross-section of EMH was assumed, with hyphal thickness equaling hyphal diameter. Based on the diameters of EMH from the six litter bags that were not incubated, two diameter classes that contained similarly large amounts of EMH litter were defined, fine (< 4 μm diameter) and coarse EMH litter (≥4 μm diameter). The amounts of fine and coarse diameter EMH were calculated by subtracting the respective amounts in control bags from those in the litter bags. HLDs of fine and coarse EMH litter (mm g−1 dry sand) were calculated from the relative frequencies of hyphae with fine and coarse diameters and the total HLD of the EMH litter. 2.7. Modeling of hyphal decomposition The remaining HLD after incubation was calculated as the fraction (%) of the mean HLD determined in the six litter bags that were not incubated. The change of the decomposition rate of the EMH litter over time was assessed by fitting decomposition models to the data of the EMH litter fractions. Decomposition models were based on a generic form of the exponential decay function (Eq. (1) in the Introduction section) proposed by Rovira and Rovira (2010): t

EMHt = EMH0 e 4

f (t )dt 0

(2)

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where EMHt is the fraction of EMH (%) remaining at time t (months) relative to the initial amount EMH0 (100%), and f(t) (month−1) describes the decomposition rate as a function of time. Thereby, f(t) relates the instantaneous change of EMHt at time t (% month−1) to EMHt (%) in the linear first-order differential equation dEMHt /dt = f (t ) EMHt (Rovira and Rovira, 2010). A lower decomposition rate indicates a slower decomposition. Decomposition rates are reported in a wide range of studies to quantify and compare decomposition processes (see for instance Berg and McClaugherty, 2003; Fernandez and Koide, 2014; Li and King, 2018; Manzoni et al., 2012; Osawa and Aizawa, 2012; Rovira and Rovira, 2010; Zhang et al., 2008). The constant-k model assumed a decomposition rate k (month−1) that is constant over time. Hence f (t ) = k which is integrated over t (Abramowitz, 1972) to

EMHt = EMH0 e

kt

t

0

For maximum decomposition rates kmax, the minimum half-life thalf(min) (months), the half-life of the most labile component of the EMH litter, was calculated in line with Eq. (10):

thalf (min) =

thalf (max ) =

(1 revisited)

(3) −1

where kmax is the maximum decomposition rate (month ) and mk is the decay rate of the decomposition rate (month−1). f (t ) was integrated over t (Abramowitz, 1972; Zucker, 1972) to t

kmax / mk (e

f (t )dt =

1)

mk t

(4)

0

and inserted into Eq. 2

EMHt = EMH0

(5)

kmin) e

(6)

mk t

which was integrated over t (Abramowitz, 1972; Zucker, 1972) to

(kmax

f (t )dt = kmin t

kmin ) mk

0

(e

mk t

1)

(7)

and inserted into Eq. 2

EMHt = EMH0 e

kmin t

(kmax kmin ) (e mk t 1) mk

(8)

To illustrate a potential change in the decomposition rate over time, irrespective of any decomposition model, constant decomposition rates ki-j were calculated between any two subsequent collections of ingrowth bags:

ki

j

=

ln(EMHj /EMHi ) tj

(9)

ti

where EMHi and EMHj is the mean fraction of EMH (%) at time ti and tj (months; ti < tj), respectively. To ease interpretation of reported decomposition rates, corresponding half-lives, the times until 50% decomposition loss, were reported (Kuzyakov et al., 2007; Mackensen et al., 2003). For constant decomposition rates k, the half-life thalf (months) was calculated after solving Eq. (1) for t:

thalf =

ln(0.5)/kmin

(12)

The programming language R (R Core Team, 2017) in combination with the R packages nlme (Pinheiro et al., 2018) and emmeans (Lenth, 2018) was used for statistical analyses. A significance level of 0.05 was chosen for all statistical tests. Correlation between the HLD of EMH litter and hyphal loss during hyphal extraction was tested by Pearson's product-moment correlation (function cor.test). Parameters for the estimation of hyphal loss during hyphal extraction from HLD of EMH litter were computed by linear regression (function lm). Mean HLDs in control and in-growth bags were compared using mixed-model one-way analysis of variance (ANOVA) with ‘incubation time’ nested within ‘season of incubation’ as a random effect (function lme). Overall mean HLDs in control and in-growth bags and overall mean hyphal diameters in litter and control bags were computed as least square mean, accounting for ‘incubation time’ nested within ‘season of incubation’ as a random effect (functions lme, emmeans). Mean HLD of EMH litter and mean HLD in control bags were compared between the pairs of bags with and without a surrounding in-growth bag using one-way ANOVA (function aov). For ANOVA comparisons, numerical variables were transformed by taking their natural logarithms to improve homogeneity of residual variances (confirmed with functions resid, plot) and to make the distribution of the residuals more normal (confirmed with functions resid, qqnorm, qqline). Decomposition models were fitted to EMH litter fractions using generalized least squares (function gnls). Occasionally observed heterogeneity of residual variances between incubation periods (functions resid, plot) was accounted for in all models (argument weights with varIdent). That the distribution of the residuals was near to the normal distribution was verified using the resid, qqnorm, and qqline functions. Variation of model parameters between diameter classes or seasons was tested by comparing models with constant or varying parameters (argument params). A decomposition model was considered valid, if all its parameters were significant (t statistics, function summary). The relative goodness of model fits was assessed with the corrected Akaike's Information Criterion (AICc) recommended for small sample sizes and/ or large numbers of parameters (function AIC; Anderson, 2008; Rovira and Rovira, 2010).

The decreasing-k model with kmin assumed a decomposition rate that exponentially decreases to the minimum kmin (> 0; month−1) over time. Such an HLD decrease function was suggested by various multiannual data sets of litter decomposition (Rovira and Rovira, 2010). A decomposition rate that approaches zero is, indeed, unlikely, if access by decomposers to the litter is not completely prevented (Olson, 1963), or, if decomposition is not prevented by harsh environmental conditions (Wieder and Lang, 1982). The decomposition rate was described as

t

ln(EMHt / EMH0 )/ kmin =

2.8. Statistical analysis

m t e kmax / mk (e k 1)

f (t ) = kmin + (kmax

(11)

The constant-k model, the decreasing-k model, and the decreasing-k model with kmin were compared for their goodness of fit to EMH litter fractions before and after incubation for 0.5–8 months from April (n = 56). Using the best-fitting decomposition model, the variation of decomposition rate parameters was assessed. Variation of parameters between the hyphal diameter classes was assessed based on the EMH litter fractions of fine and coarse EMH before and after incubation for 0.5–8 months from April (n = 112). Variation in the decomposition parameters between seasons of in-growth bag incubation was assessed, using the EMH litter fractions after incubation for 0.5–8 months from April, August, October, and February (n = 83). Due to short incubation periods, variation of kmax and mk but not kmin could be assessed among seasons of in-growth bag incubation. kmin was preset according to the outcomes of the initial model comparison. The collected and herein analyzed EMH litter data is openly accessible (Schäfer et al., 2019).

f (t )dt = kt and inserted in Eq. (1):

mk t

ln(0.5)/kmax

For minimum decomposition rates kmin, the maximum half-life thalf(max) (months), the half-life of the most recalcitrant component of the EMH litter, was calculated in line with Eqs. 10 and 11:

The decreasing-k model assumed a continuous, exponentially decreasing decomposition rate over time as suggested by Li and King (2018) for dead EMH in in-growth bags:

f (t ) = kmax e

ln(EMHt / EMH0 )/ kmax =

ln(EMHt /EMH0 )/ k =

ln(0.5)/ k

(10) 5

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3. Results

for 0.5, 1, and 2 months from April, August, October, and February (n = 83), several models converged and had significant parameters (t statistics, p < 0.001; Table 1). The best fit was found for a model with different kmax for spring-summer (April and August) and for autumnwinter (October and February) and a constant mk (Table 1; Fig. 5a and b). From the kmax of the best-fitting model, minimum half-lives of 0.26 ± 0.04 for spring-summer and 0.61 ± 0.09 months for autumnwinter were estimated.

The mean hyphal length density (HLD) of extraradical mycorrhizal fungal hyphae (EMH) before in-growth bag incubation was 1082 ± 41 mm g−1 dry sand (n = 6; ± standard error). HLD of EMH litter remaining in litter bags and hyphal loss during hyphal extraction were significantly negatively correlated (n = 16; Pearson's productmoment correlation, Pearson's r = −0.66, t = −3.30, p = 0.005). The linear regression line between HLD of EMH litter and hyphal loss (R2 = 0.44, p = 0.005) had an intercept of 13.5 ± 2.5% (t = 5.40, p < 0.001) and a slope of −0.017 ± 0.005% mm−1 g dry sand (t = −3.30, p = 0.005). Mean HLD was significantly lower in control bags (93 ± 25 mm g−1 dry sand) compared to in-growth bags (133 ± 25 mm g−1 dry sand, n = 91 each; least square means; mixedmodel ANOVA, F = 12.80, p < 0.001). Mean HLD of EMH litter was not significantly different between litter bags with (336 ± 34 mm g−1 dry sand) and without surrounding in-growth bags (244 ± 48 mm g−1 dry sand) after two months of incubation (n = 10 each; one-way ANOVA, F = 0.36, p = 0.558). Mean HLD was, however, significantly lower for control bags with (108 ± 24 mm g−1 dry sand) than without a surrounding in-growth bag (275 ± 58 mm g−1 dry sand; one-way ANOVA, F = 10.99, p = 0.004). The mean hyphal diameter was 4.4 ± 0.4 μm (n = 11917) and 4.2 ± 0.4 μm (n = 2814) in litter and control bags, respectively (least square means). The constant-k model, the decreasing-k model, and the decreasing-k model with kmin were compared for their goodness of fit to EMH litter fractions before and after incubation for 0.5–8 months from April (n = 56). In all decomposition models, parameters were significant in all cases (t statistics, p < 0.049). The best fit was found for the decreasing-k model with kmin (Table 1; Fig. 3a and b). From the maximum (kmax) and minimum decomposition rate (kmin) of the decreasing-k model with kmin a minimum half-life of 0.28 ± 0.08 months and a maximum half-life of 6.9 ± 3.4 months were estimated. When the decreasing-k model with kmin was fitted to fine and coarse EMH litter fractions before and after incubation for 0.5–8 months from April (n = 112), only a model that had different kmax for fine and coarse EMH but otherwise constant parameters converged and showed significant parameter estimates (t statistics, p < 0.030; Table 1; Fig. 4a and b). From the kmax of fine and coarse EMH a minimum half-life of 0.42 ± 0.11 and 0.22 ± 0.06 months was estimated, respectively. When the decreasingk model with kmin was fitted to the EMH litter fractions after incubation

4. Discussion Hyphal in-growth into control bags, and by inference litter bags of the same design, was not blocked but significantly reduced by 30% compared to the in-growth bags as a consequence of the 1 μm mesh. The mean hyphal diameter in control bags was 4.2 μm, which suggests that hyphal diameters are plastic and allow hyphae to pass 1 μm mesh. Indeed, reduced hyphal in-growth into 1 μm mesh has been observed for extraradical mycorrhizal fungal hyphae (EMH) of ectomycorrhizal (ECM; Teste et al., 2006; Heinemeyer et al., 2012) and arbuscular mycorrhizal (AM) fungi (Nottingham et al., 2010), although the diameter of EMH commonly exceeds 2 μm (Friese and Allen, 1991; Leake et al., 2004; Smith and Read, 2008). Omission of the surrounding ingrowth bag significantly increased the HLD in control bags by 155%, which is in line with previous observations that filling in-growth bags with sand has a negative effect on EMH penetration and biomass formation that increases with bag size (Hendricks et al., 2016; Mikusinska et al., 2013). However, omission of the surrounding in-growth bag did not significantly reduce the HLD of EMH litter (the difference in HLD between the litter and control bags), indicating that the in-growth bag environment may have had a limited negative effect on EMH decomposition and that dynamics in the HLD of EMH litter reflected EMH decomposition in the soil. The decomposition of field-grown EMH litter over the course of eight months of in-growth bag incubation was best explained by the decreasing-k model with kmin which assumed an exponential decrease of the decomposition rate over time from a maximum (2.5 month−1; kmax) to a minimum (0.1 month−1; kmin; Table 1; Fig. 3a and b). kmax and kmin corresponded to a minimum and maximum half-life of 0.3 and 7 months, respectively. A drop of the decomposition rate over time agrees with previous reports for many types of litter, including litter of mycorrhizal fungal fruit bodies and laboratory-grown EMH in forest soil

Table 1 Decomposition models fitted to the litter fractions (% of initial mean hyphal length density) of extraradical mycorrhizal fungal hyphae (EMH) in in-growth mesh bags incubated in the soil of a Chamaecyparis obtusa plantation. Decomposition models were fitted using generalized least squares. Model parameters are represented as estimates ± standard errors. Only decomposition models that had significant parameters in all cases are listed (t statistics; significance level 0.05). For two-month incubations, kmin was preset according to model M3. The relative goodness of model fit is indicated by the corrected Akaike's Information Criterion (AICc). For definitions of model types and their parameters see the Materials and Methods section. Model name

Model type

Start of incubation

Model type comparison (n = 56) M1 Constant-k April M2 Decreasing-k April M3 Decreasing-k with kmin April EMH diameter class comparison (n = 112) M4 Decreasing-k with kmin April “ “ “ Seasonal comparison (n = 83) M5 Decreasing-k with kmin April, August, October, February M6 Decreasing-k with kmin April, August “ “ October, February M7 Decreasing-k with kmin April “ “ August “ “ October “ “ February

Incubation time (months)

EMH diameter

8 8 8

Parameter (month−1)

AICc

k

kmax

mk

kmin

all all all

0.72 ± 0.04 – –

– 1.9 ± 0.3 2.5 ± 0.7

– 1.2 ± 0.3 2.2 ± 0.9

– – 0.10 ± 0.05

472.7 445.3 443.9

8 “

fine coarse

– –

1.6 ± 0.4 3.1 ± 0.9

2.0 ± 0.8 “

0.09 ± 0.04 “

954.8 “

2

all

–

1.5 ± 0.3

1.6 ± 0.4

0.1 (preset)

709.7

2 “ 2 “ “ “

all “ all “ “ “

– – – – – –

2.7 1.1 2.5 2.8 1.2 1.0

2.2 ± 0.5 “ 2.1 ± 0.5 “ “ “

0.1 (preset) “ 0.1 (preset) “ “ “

637.6 “ 639.8 “ “ “

6

± ± ± ± ± ±

0.4 0.2 0.4 0.4 0.2 0.2

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Fig. 3. Remaining litter fractions of extraradical mycorrhizal fungal hyphae (EMH) in in-growth bags (n = 56) after incubation for 0.5–8 months in a Chamaecyparis obtusa plantation (a) and corresponding decomposition rates (b). The remaining EMH litter fractions (a) are shown as means (dots) with standard errors (whiskers). Constant decomposition rates ki-j between two consecutive collection dates ti and tj (b) are shown as white bars. Model fits (a, b) of the constant-k model (model M1), decreasing-k model (model M2), and decreasing-k model with kmin (model M3; Table 1) are shown as lines.

Fig. 4. Remaining fractions of fine (diameter < 4 μm) and coarse (diameter ≥ 4 μm) extraradical mycorrhizal fungal hyphae (EMH) in in-growth bags (n = 112) after incubation for 0.5–8 months in a Chamaecyparis obtusa plantation (a) and corresponding decomposition rates (b). The remaining EMH litter fractions (a) are shown as means (dots) with standard errors (whiskers). Constant decomposition rates ki-j between two consecutive collection dates ti and tj (b) are shown as bars with colors corresponding to dot colors. The fit of model M4 (Table 1) with different maximum decomposition rates kmax for fine and coarse EMH litter is shown as lines (a, b).

(Brabcová et al., 2016; Fernandez and Koide, 2014; Li and King, 2018; Rovira and Rovira, 2010). Good description of the decomposition process by a model with an exponentially decreasing decomposition rate agrees with reports of several studies that compared various models of decomposition (Carpenter, 1982; Ezcurra and Becerra, 1987; Godshalk and Wetzel, 1978; Manzoni et al., 2012; Rovira and Rovira, 2010). For dead EMH in in-growth bags, Li and King (2018) inferred an exponential decrease of the decomposition rate over time based on the three-month decomposition process of laboratory-grown EMH in forest soil (Fernandez and Koide, 2014). Our analysis of decomposition over the course of eight months of field-grown EMH in in-growth bags supports their findings. However, the significance of parameter kmin and the best fit of the decreasing-k model with kmin (Table 1; Fig. 3a) suggest an exponential decrease of the decomposition rate to a minimum rather than to zero as Li and King (2018) suggested. Estimation of EMH decomposition with in-growth bags may, hence, require long-term decomposition data to specify kmin. Indeed, model outcomes in Li and King (2018) were reported to be less reliable for long than short incubation periods. Compared between EMH diameter classes, kmax was significantly lower for fine (1.6 month−1; diameter < 4 μm) compared to coarse

EMH litter (3.1 month−1; diameter ≥ 4 μm; Table 1; Fig. 4a and b). kmax for fine and coarse EMH corresponded to a minimum half-life of 0.4 and 0.2 months, respectively. A lower decomposition rate for fine compared to coarse EMH litter contradicts the, so far unconfirmed but intuitive, assumption that fine EMH decompose more rapidly than coarse EMH (Langley and Hungate, 2003; Steinberg and Rillig, 2003). A smaller EMH diameter results in a higher surface area exposed to decomposers per hyphal volume and a smaller hyphal volume per HLD to be decomposed. EMH in different diameter classes may, however, be associated with different branching orders and functions within an EMH network (Friese and Allen, 1991) or with different AM fungal species (Leake et al., 2004). Effects of functional and species-related differences in EMH structure and chemical composition may thus be more important than hyphal diameter. Similar to our observation, coarser roots decomposed faster than finer ones (Langley and Hungate [2003] and references therein) and coarser rhizomorphs, densely bundled EMH of ECM fungi, decomposed faster than finer ones (Fernandez et al., 2016; McCormack et al., 2010), indicating that the observed phenomenon is not limited to EMH litter. Compared between seasons of in-growth bag incubation, kmax differed. It was significantly lower for autumn-winter (October and 7

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fungi (Leifheit et al., 2015) may explain a lower kmax at higher soil temperatures and times of active plant growth. It was shown that soil water moved within minutes through a fine mesh of 0.5 μm mesh size (Teste et al., 2009), suggesting mineral nutrient flow and bacterial migration between the litter bags and the surrounding soil. Hence, potential negative effects on bacterial activity inside litter bags by competition with roots and EMH in the surrounding soil was possible. A rapid decrease of the decomposition rate of the field-grown EMH litter over time was indicated in all observed decomposition processes regardless of the EMH diameter class or season of in-growth bag incubation (Figs. 3b, 4b and 5b). Loss in HLD of EMH litter during the first month of in-growth bag incubation (mean losses of 40–78% month−1; Figs. 3a, 4a and 5a) was similar to the large initial dry mass loss of laboratory-grown EMH litter in soil (20–60% month−1; Fernandez and Koide, 2014, 2012). Estimated kmin (0.09–0.10 month−1; maximum half-life of 6.9–7.4 months), which was reached after 2–4 months of incubation (Figs. 3b, 4b and 5b), was similar to the constant decomposition rates estimated for dead EMH in in-growth bags incubated for 4.5–29 months in a warm-temperate Pinus taeda stand (0.11–0.13 month−1 [Ekblad et al., 2016]; half-life of 5.4–6.6 months). Our results, therefore, indicate that the timing of observation may explain the reported differences in EMH decomposition dynamics between laboratory-grown EMH and field-grown EMH. To understand how EMH regulate the cycling and retention of plantassimilated C in forest soils, recent studies proposed the estimation of EMH production, mortality, and decomposition with mass-balance models combined with in-growth bags (Ekblad et al., 2016; Hagenbo et al., 2017; Li and King, 2018). Our results point at profound differences in the decomposition rates of field-grown EMH between the initial and later stages of the decomposition and between seasons which requires observation in the field and consideration in mass-balance models. Observation may be achieved by frequent incubation of EMH litter bags, consideration in mass-balance models by introducing cohorts of decomposing EMH that differ in the time of EMH death and time-dependent decomposition rates used by Li and King (2018). However, this study is not a comprehensive review of previously suggested mass-balance models which differ in more than their assumptions on EMH decomposition and must be directly compared for their applicability and outcomes with accumulating field data. Furthermore, we assessed EMH decomposition by means of HLD measurements. To confirm our results, tracking of the full mineralization process of EMH in in-growth bags by measurement of CO2 efflux or C masses is required. Lastly, analysis of fungal group-specific lipid fatty acids would have helped to determine fractions of different fungal groups in the field-grown EMH litter (Nilsson et al., 2005). Contribution of non-mycorrhizal fungal hyphae and EMH of multiple mycorrhizal types to EMH litter grown under field conditions is unavoidable and needs further investigation in future studies. EMH of AM fungi were suggested to decompose more rapidly than EMH of ECM fungi (Langley and Hungate, 2003). While the similarity of initial decomposition losses between our study and former studies with laboratory-grown EMH of ECM fungi (Fernandez and Koide, 2014, 2012) indicates a limited influence of the mycorrhizal type, direct comparison of EMH decomposition between AM and ECM fungi are warranted to confirm this suggestion.

Fig. 5. Remaining litter fractions of extraradical mycorrhizal fungal hyphae (EMH) in in-growth bags (n = 83) after incubation for 0.5–2 months in a Chamaecyparis obtusa plantation in four different seasons (a) and corresponding decomposition rates (b). The remaining EMH litter fractions (a) are shown as means with standard errors as whiskers. Constant decomposition rates ki-j between two consecutive collection dates ti and tj (b) are shown as bars with colors corresponding to those of the dots. The fit of model M6 (Table 1) with different maximum decomposition rates kmax for the April and August and October and February dataset is shown as lines.

February; 1.1 month−1) than spring-summer (April and August; 2.7 month−1) in the best fitting decomposition model (Table 1; Fig. 5a and b). kmax for autumn-winter and kmax for spring-summer corresponded to a minimum half-life of 0.6 and 0.3 months, respectively. Daily mean soil water content was similar within 0.5 months from the start of each insertion, showing minima of 0.13–0.16 m3 m−3 and maxima of 0.19–0.24 m3 m−3 (Fig. 1). Daily mean soil temperature, on the contrary, reached its overall maximum (25.1 °C) and minimum (0.5 °C) soon after the start of incubations from August and from February, respectively (Fig. 1) and, hence, was likely responsible for the difference in kmax between the former and latter. An effect of ambient temperature on the decomposition rate has been reported for many types of litter (Zhang et al., [2008] and references therein). A higher kmax for April compared to October despite lower soil temperatures within 0.5 months from the start of incubation (Fig. 1) may have resulted from increased activity of microbial decomposers in the soil surrounding ingrowth bags in spring. Leaf litter fall (Inagaki et al., 2008) and fine root mortality in in-growth cores (Osawa and Aizawa, 2012) were reported to increase during winter months in warm-temperate C. obtusa plantations. Soil litter input, in turn, was an important driver of the activity of microbial decomposers in a nearby deciduous–evergreen forest (Kominami et al., 2012), suggesting co-metabolization to be important for hyphal decomposition. Furthermore, bacterial mineral nutrient limitation due to competition with plants (Barel et al., 2019) and AM

Declarations of interest None. Acknowledgments The authors thank Jérôme Bertrand for assistance with fieldwork and Yoshiko Kosugi for providing access to the Kiryu Experimental Watershed, her advice on the plot location, and comments on the manuscript. This work was supported by the Sasakawa Scientific 8

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Research Grant from The Japan Science Society [grant number 29–516]. The funding body was not involved in the study design, data collection, analysis, and interpretation, report writing, and the decision to submit the article for publication. Additional information: Professor Akira Osawa, coauthor of this article, died in May 2019 during revision of this article.

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