ORIGINAL ARTICLE

Y. Fukasawa • O. Tateno • Y. Hagiwara

D. Hirose • T. Osono

Fungal succession and decomposition of beech cupule litter

Received: 31 October 2011 / Accepted: 28 March 2012 / Published online: 26 April 2012� The Ecological Society of Japan 2012

Abstract Beech cupule litter is the second largest (next toleaf litter) component of total annual litterfall in mastyears, and makes an important contribution to carbonbudgets in beech forest soils. We investigated thedecomposition processes of beech cupule litter over a30-month period with reference to the role of fungalsuccession in the decomposition of acid-unhydrolyzableresidue (AUR) and holocellulose. During the studyperiod, weight loss of holocellulose occurred, while therewas little weight loss of AUR, and 77 % of the originalcupule weight remained at the end of the study period.Xylaria sp.1, Geniculosporium sp. and Nigrospora sp.that can attack holocellulose selectively caused mass lossof holocellulose and were responsible for the cupuleweight loss. Although the beech cupule is a woodyphyllome and its lignocellulose composition is similar tothat of coarse woody debris (CWD) rather than leaflitter of beech, the selective decomposition of holocel-lulose by fungi was similar to the decay process of leaflitter rather than CWD.

Keywords Fagus crenata Æ Fungi Æ Lignin ÆLignocellulose Æ Xylaria

Introduction

Litter decomposition is an important process control-ling soil organic matter formation in forest ecosystems(Swift et al. 1979). Forest litter includes various treetissues such as leaves, flowers, fine woody debris(FWD: twigs, branches, and cupules), coarse woodydebris (CWD: boles, stems, and coarse roots), and fineroots. The extent to which each of them is quantita-tively important differs among forests (Vogt et al.1986). In beech forests, cupule (woody organ coveringseeds) litter is quantitatively important. Mast seeding(masting) of Japanese beech (Fagus crenata Blume)occurs every 5–7 years (Kashimura 1952; Kikuchi 1968;Maeda 1988; Hiroki and Matsubara 1995; Suzuki et al.2005). Cupule litter is the second largest componentand constitutes up to 28 % of total annual litterfall inmast years (Kawada and Maruyama 1986). Thus, it isimportant to clarify the cupule decomposition processin order to better understand carbon cycling in beechforests.

Cupule tissue is a woody phyllome and is composedof highly recalcitrant lignocellulose complex. Fungi playa central role in lignocellulose decomposition in forestecosystems because they secret extracellular enzymesthat catalyze decomposition of acid-unhydrolyzableresidue (AUR, formerly referred to as lignin) andholocellulose polymers (Eriksson et al. 1990). Fungalspecies with various decay abilities successively colonizecupule litter and consequently promote the decay pro-cess (Rayner and Boddy 1988). Carre (1964) conducted a2-year survey of the decomposition of cupule litter ofEuropean beech (F. sylvatica L.) and fungal fruitingbodies occurring on it. He found negligible weight loss(6 %) during the 2-year period, and concluded that theoccurrence pattern of fungal fruiting bodies was morestrongly affected by season than the decay process.However, occurrences of fruiting bodies are poor indexfor fungal succession inside the substrates (Rayner andBoddy 1988). Thus, research including fungal isolation

Y. Fukasawa (&)Laboratory of Forest Ecology, Graduate Schoolof Agricultural Science, Tohoku University,Naruko-onsen, Osaki, Miyagi 989-6711, JapanE-mail: fukasawayuu@gmail.comTel.: +81-229-847397Fax: +81-229-846490

O. Tateno Æ Y. HagiwaraGraduate School of Agriculture, Kyoto University,Kyoto 606-8502, Japan

D. HiroseCollege of Pharmacy, Nihon University, Funabashi,Chiba 274-8555, Japan

T. OsonoCenter for Ecological Research, Kyoto University,Otsu, Shiga 520-2113, Japan

Ecol Res (2012) 27: 735–743DOI 10.1007/s11284-012-0947-3

should be conducted to better understand the fungaldecomposition of beech cupule litter.

Recently, we conducted comprehensive surveys offungal decomposition of Japanese beech litters such asleaf litter (Osono andTakeda 2001) andCWD(Fukasawaet al. 2009a, b). Those studies found differences in thefungal community structure and consequent decay pro-cess of lignocellulose between leaf and woody litters. Inbeech cupule litter, previous studies found some spe-cialized fungal species such as Dasyscyphella longistipi-tata Hosoya (Ono and Hosoya 2001) and Xylariacarpophila (Pers.) Fr. (Ellis and Ellis 1997). Thus, it ishypothesized that the fungal community and decompo-sition process of beech cupule litter differ from those ofbeech leaf litter and CWD. The aims of the present studywere (1) to find relationships between the cupuledecomposition process and fungal succession; and (2) tocompare cupule decay abilities of various fungi isolatedfrom beech cupule litter. Based on the results, we de-scribe the fungal decomposition process of beech cupulelitter and discuss its differences with the decompositionprocesses of leaf litter and CWD.

Materials and methods

Study site

The study was carried out in Ashiu Experimental Forestof Kyoto University (35�18¢N and 135�43¢E), Kyoto,Japan. Over the past 29 years, the mean annual tem-perature was 11.7 �C and the mean monthly tempera-ture ranged from 0.4 �C in January to 25.5 �C in Augustat the office of Ashiu Experimental Forest about 5 kmfrom the study site. The mean annual precipitation overthe past 29 years was 2,353 mm. The study area iscovered with snow from December to April. The studysite (altitude 660 m) is located in a mountainous area ofa cool temperate natural forest dominated by F. crenataand Quercus crispula Bl. Vegetation of the study site wasdescribed in Tateno and Takeda (2003). The site hasbeen intact since at least 1898. A study plot 20 · 10 m inarea was laid out on the lower part of a northwest-facingslope and was divided into ten subplots of 4 · 5 m forthe litterbag experiment.

Litterbag experiment

In the study site, mass flowering of F. crenata and massproduction of cupules were observed in 2005, and a largeamount of cupules fell on the forest floor during theautumn. Cupules were collected in April 2006 from theforest floor and taken to the laboratory. Thus, cupuleshave passed a winter covered with snow before collec-tion. The decomposition processes of fallen cupules ofF. crenata were studied using the litterbag method(Crossley and Hoglund 1962). Cupules were air-dried atroom temperature (approx. 15–20 �C) for 1 week.

Cupules (3 g, 7–10 cupules per litterbag) were enclosedin a litterbag (15 · 15 cm) made of polypropylene shadecloth with a mesh size of approx. 2 mm. A total of 180bags were prepared. Twenty bags were used for thedetermination of oven-dry mass at 40 �C, fungal isola-tion and observation, and chemical analysis of initialmaterials, and the other 160 bags were set on the forestfloor for the further decomposition study.

The decomposition study covered a 30-month periodfrom May 2006 to November 2008. Sixteen litterbagswere placed on the surface of the forest floor in each often subplots in May 2006. The bags were attached to theforest floor with metal pins to prevent movement or lossand to ensure good contact between the bags and litterlayer. Sampling of the bags took place eight times,namely, at 3 (Aug 2006), 6 (November 2006), 12 (May2007), 15 (August 2007), 18 (November 2007), 24 (May2008), 27 (August 2008), and 30 months (November2008). On each sampling occasion, 20 bags were re-trieved from ten subplots, two bags per subplot. Thebags were placed in paper bags and taken to the labo-ratory. Foreign plant remains attached to the outside ofthe bags were carefully removed with forceps. Ten of the20 bags were used for fungal isolation and observationand the other ten for chemical analysis.

Isolation and observation of fungi

A surface disinfection method was used for the isolationof fungi from decomposing cupules. Fungal isolationwas carried out within 24 h of sampling. One cupule wastaken arbitrarily from each of the ten bags for fungalexamination and cut into four equivalent pieces and onestem. Thus, a total of 40 pieces and ten stems were usedfor each sampling. Methods of surface-disinfection weredescribed in Osono et al. (2008), except that surface-disinfected cupules were inoculated onto 2 % malt ex-tract agar [malt extract 2.0 %, agar 20.0 % (w/v)] andincubated at 20 �C for 8 weeks. Identification of fungalisolates was based on micromorphological observations,with reference to Domsch et al. (1980) and Ellis (1971,1976). The frequency of occurrence of an individual specieswas calculated as a percentage based on the number ofcupules with the species among the ten cupules regardlessof the number of cupule pieces or stems with the species. Aspecies was regarded as frequent when the frequency ofoccurrence of the species was greater than 50 % at any ofthe nine sampling occasion. Only the results for the fre-quent species are shown in the present study.

An additional two cupules were taken arbitrarilyfrom each bag, and a total of 20 cupules were used forobservations of the incidence of fruiting bodies on eachsampling occasion. Occurrence of fruiting bodies ofDasyscyphella longistipitata and Xylaria carpophila onthe surface of cupules were observed with a binocularmicroscope at 40· magnification. The frequency of thesespecies was calculated as a percentage based on thenumber of cupules with the species among 20 cupules.

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We occasionally encountered fruiting bodies of otherascomycetes and basidiomycetes during the study peri-od, but their frequencies were very low and were notreported here.

Chemical analysis

Cupules decomposing in bags for chemical analysis wereoven-dried at 40 �C for 1 week to determine theremaining mass. Mean values of remaining mass werecalculated for each sampling and expressed as a per-centage of the original mass. The decomposition rate ofcupules in the field was calculated using Olson’s k (Olson1963) according to the following equation:

Wt ¼ W0 � exp �ktð Þ

where Wt is the cupule weight after a given period, W0 isthe original cupule weight, k is the decomposition rate,and t is the time in years.

The cupules from ten bags were combined to makeone sample, which was ground in a laboratory mill andpassed through a 0.5-mm screen. The amount of AURwas estimated by gravimetry according to a standard-ized method using hot sulfuric acid digestion (King andHeath 1967). The total amount of carbohydrates wasestimated by means of the phenol–sulfuric acid method(Dubois et al. 1956). Soluble carbohydrates were ex-tracted with 50 % methanol and their content wasmeasured with the phenol–sulfuric acid method. Theholocellulose fraction was calculated as the differencebetween the total carbohydrates and the soluble carbo-hydrates. Lignocellulose index (LCI) was calculated asan indicator of the relative availability of carbon energysources in the litter according to the following equation:

LCI ¼ content of holocellulose/ðcontent of holocelluloseþ content of AURÞ

Pure culture decomposition test

The ability of 18 isolates of ten fungal taxa that occurredfrequently on beech cupules (Table 1) to causemass loss ofcupules and of AUR and total carbohydrates in cupuleswas studied with pure culture decomposition test accord-ing to the method described in Osono and Hirose (2009).Cupules used in the tests were collected in April 2006 fromthe forest floor of the study site and used in the pure culturetest. In summary, one cupule (371 mg on average) wassterilized by exposure to ethylene oxide gas for 6 h, placedon the surface of a Petri dish (9 cm in diameter) containing20 ml of 2 % agar, inoculated with fungi, and incubatedfor 12 weeks at 20 �C in darkness. After incubation, theinoculated cupules were retrieved, oven-dried at 40 �C for1 week, and weighed. The initial, undecomposedmaterialswere also sterilized, oven-dried at 40 �C for 1 week, andweighed to determine the original mass. Four plates wereprepared for each isolate, and four uninoculated, incu-bated plates served as a control. Mass loss of a cupuleinoculated with each of fungal strain was determined as apercentage of the original mass, subtracting the mass lossin control. Chemical analyses were performed for cupulesinoculatedwith those fungal isolates that causedmore than5 % loss of cupule mass. Contents of AUR and totalcarbohydrates of initial, control, and inoculated cupuleswere determined according to the methods describedabove, and mass losses of AUR and total carbohydrateswere determined and expressed as a percentage of theoriginal masses. AUR/carbohydrates loss ratio (AUR/C)

Table 1 Mass loss (% original mass) of cupules and organic chemical components and AUR/carbohydrate mass loss ratio (AUR/C)caused by fungi under pure culture condition

Fungus Code Mass loss AUR/C

Total mass AUR Total carbohydrates

Geniculosporium sp. Ge5 12.7 ± 0.8 7 27.7 0.25Geniculosporium sp. Ge6 10.4 ± 0.9 8.7 17.4 0.50Geniculosporium sp. Ge8 9.1 ± 0.4 5.2 19.5 0.27Xylaria sp.1 Xy1 10.4 ± 1.2 4.2 20.3 0.21Xylaria sp.1 Xy7 8.9 ± 0.9 �0.3 22.2 �0.01Xylaria sp.1 Xy8 8.2 ± 0.7 1.6 16.6 0.10Xylaria carpophila Xc8 5.6 ± 0.8 4.1 6.2 0.66Nigrospora sp. Ni2 10.4 ± 0.6 0.1 22.9 0Nigrospora sp. Ni5 0.9 ± 0.5 nd nd ndPhomopsis sp. Ph3 5.2 ± 0.5 3.3 13.3 0.25Phomopsis sp. Ph1 3.9 ± 0.4 nd nd ndAscochyta fagi As0 2.3 ± 0.4 nd nd ndTrichoderma sp. Tr7 2.0 ± 0.3 nd nd ndTrichoderma sp. Tr6 1.6 ± 0.5 nd nd ndEpicoccum nigrum Ep5 1.3 ± 0.2 nd nd ndDasyscyphella longistipitata Da7 0.9 ± 0.2 nd nd ndPenicillium sp. Pe2 1.3 ± 0.5 nd nd ndPenicillium sp. Pe5 �0.4 ± 0.1 nd nd nd

Values are means ± standard errors (n = 4)nd not determined, AUR acid-unhydrolyzable residue

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is a useful index of the substrate utilization pattern of eachfungal species. AUR/C of each fungal species was calcu-lated according to the equation:

AUR/C¼mass loss of AUR ð% of original AUR massÞ=mass loss of total carbohydrates

ð% of original total carbohydrate massÞ

Results

Fungal succession

Nine species were frequently isolated from decomposingcupules, and succession of these fungi was observed

during decomposition (Fig. 1). The frequency of Xylariasp.1 fluctuated at 90–100 % as the decomposition pro-gressed. The frequencies of Phomopsis spp., Ascochytafagi, and Epicoccum nigrum were between 60–70 % ini-tially, decreased thereafter, and finally fell to 0 % withinthe study period. The frequency of Trichoderma spp.increased to 80 % during the first 6 months and thenfluctuated at 60–90 % as the decomposition progressed.Similarly, the frequencies of Geniculosporium spp., whitesterile 1, Nigrospora spp., and Penicillium spp. wereinitially low and then increased at 6, 18, 24, and/or30 months of decomposition.

The frequency of fruiting bodies of Dasyscyphellalongistipitata increased in May of the second and thethird years (i.e., at 12 and 24 months of decomposition,respectively) (Fig. 2). Fruiting bodies of Xylaria

50

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Xylaria sp.1

Phomopsis spp.

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75Ascochyta fagi

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75Trichoderma spp.

Epicoccum nigrum

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Geniculosporium spp.

Nigrospora spp.

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Penicillium sppWhite sterile 1

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Penicillium spp.White sterile 1

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Fig. 1 Frequencies of fungiisolated from cupules versustime during decomposition(n = 10)

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carpophila were absent from cupules between 0 and12 months of decomposition and increased thereafter,with a frequency of occurrence of 10–20 % (Fig. 2).

Chemical changes

The mass loss of cupule litter was linear with respect tothe duration of decomposition, and 77 % of the originalmass remained at the end of a 30-month period (Fig. 3).The decomposition rate was 0.111 year�1 (n = 9,

R2 = 0.983). The change in the mass of AUR wasnegligible during the 30-month period, whereas the massof holocellulose decreased linearly with time, and 55 %of the original mass remained at the end of the studyperiod (Fig. 3). The mass of soluble carbohydrates re-mained nearly constant during the first 15 months andthen increased to reach 147 % of the original mass ofsoluble carbohydrates at the end of the study period(Fig. 3). The contents of AUR and soluble carbohy-drates increased, whereas that of holocellulose decreasedduring decomposition (Table 2). Consequently, LCIdecreased during decomposition (Table 2).

Decomposing ability

The loss of cupule litter mass caused by 18 isolates offungi ranged from �0.4 to 12.7 % (Table 1). Isolates ofGeniculosporium sp. caused the greatest mass loss, fol-lowed by three isolates of Xylaria sp., one isolate ofNigrospora sp. Ni2, X. carpophila Xc8, and Phomopsissp. Ph3. The other fungal isolates caused less than a 5 %loss of cupule mass. The mass loss of AUR caused bythe nine isolates that caused more than 5 % loss ofthe original cupule mass ranged from �0.3 to 8.7 %(Table 1). The three isolates of Geniculosporium sp.caused the greatest mass loss of AUR, followed byXylaria sp.1 Xy1 and X. carpophila Xc8. Two isolates ofXylaria sp.1 (Xy7 and Xy8) and Nigrospora sp. Ni2caused negligible mass loss of AUR. The mass loss oftotal carbohydrates caused by nine isolates ranged from6.2 to 27.7 % (Table 1). Geniculosporium sp.1 Ge5caused the greatest mass loss of total carbohydrates,followed by Nigrospora sp. Ni2 and Xylaria sp.1 Xy7.AUR/C of the nine isolates ranged from �0.01 to 0.66

Dasyscyphellalongistipitata

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Xylaria carpophila

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Fig. 2 Frequencies of fruiting bodies of fungi on cupules versustime during decomposition (n = 20)

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Fig. 3 Remaining mass ofcupule’s total mass and organicchemical constituents versustime during decomposition.AUR acid-unhydrolyzableresidue

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(Table 1). Xylaria carpophila Xc8 exhibited the greatestAUR/C, followed by Geniculosporium sp. Ge6.

Discussion

Patterns of beech cupule litter decomposition by fungalcolonizers

The rate of mass loss of beech cupule litter recorded inthe present study (Fig. 3) was more than 3 times fasterthan that recorded in Europe (Carre 1964), where only6 % mass loss was recorded during a 2-year period. Ingeneral, litter decay rate is mainly regulated by chemicalcomposition of litter, climate environment, and decom-poser organisms (Berg and McClaugherty 2003). Espe-cially climate has dominant effects on litter decompositionrates on such a regional scale (Meentemeyer 1984).Although mean annual temperature was not so differentbetween this study and Carre’s sites, annual precipita-tion differed greatly: annual precipitation at the presentstudy site was 2,353 mm, which was approximately threetimes higher than that at the study site of Carre (1964),where the annual precipitation was 810 mm. Climatealso affects decomposer communities and indirectlyalters decay rate. Osono (2011) founds decomposingability of species within fungal assemblages was greaterin warmer than in cooler climates.

The mass loss data of organic components suggestedthat selective decomposition of holocellulose occurredduring the 30-month study period (Fig. 3). Xylaria sp.1,which was frequently isolated from beech cupule litterduring the study period (Fig. 1), showed substantialability to promote the decay of total carbohydrate in thepure culture decay test (Table 1), and thus was suggestedto be a main decomposer of holocellulose of beech cu-pule litter. Fungi in Xylariaceae, including Xylaria, areknown to be holocellulose decomposers of wood (Mer-rill et al. 1964; Rogers 1979; Sutherland and Crawford1981; Nilsson et al. 1989; Whalley 1996; Worrall et al.1997) and leaf litter (Osono and Takeda 2002). Xylariasp.1 was also isolated from live beech cupules and twigs(Fukasawa et al. 2009c; Tateno pers. comm.). Thus,

Xylaria sp.1 is an endophytic fungus of beech cupulesthat has decay ability after litterfall of cupules.

The frequency of Trichoderma spp. increased duringdecomposition (Fig. 2). Species in the genus Tricho-derma are also known to be holocellulose decomposers(Nilsson 1973; Domsch et al. 1980; Song et al. 2010).Although their decay abilities for wood are relativelyweak, these fungi would be stimulated after delignifica-tion by prior decomposers (Tanaka et al. 1988; Fukas-awa et al. 2011). However, in the present study, theircontributions to holocellulose decomposition are dubi-ous because their counterpart, Xylaria sp.1, was not anactive AUR decomposer and thus AUR content re-mained high in cupule litter. Wood decay ability maynot be so important for the nutrition of the fungi in sucha case because Trichoderma spp. have a more or lessmycoparasitic nature (Dennis and Webster 1971; Kumaret al. 2010). The pure culture decay test also suggestedthat the abilities of Trichoderma spp. to decay beechcupule litter were clearly low (Table 1).

Three fungi quickly disappeared in the early stage ofcupule decomposition, namely, Phomopsis sp., Asco-chyta fagi, and Epicoccum nigrum (Fig. 1), which werealso isolated from live beech cupules (Tateno pers.comm.), and thus suggested to be endophytes. Theircontributions to cupule decomposition may be smallbecause little weight loss was recorded in pure culturedecay tests (Table 1).Geniculosporium sp. andNigrosporasp. caused substantial weight loss of total carbohydrate,and may contribute to beech cupule decomposition. Thecupule decay ability of Penicillium sp. was low (Table 1).This species may be a secondary sugar fungus, as de-scribed by Hudson (1968), utilizing low molecular weightsoluble sugars such as glucose released from the holocel-lulose decay process (Fig. 3).

Two species recorded as fruiting bodies, Xylariacarpophila and Dasyscyphella longistipitata, showedlow decay abilities (Table 1). Although X. carpophiladecomposed AUR at a similar rate to total carbohydrate(Table 1), their contributions to the cupule decay pro-cess may have been small during the study period, as theAUR weight loss was negligible (Fig. 3). This specieswas distinguished from Xylaria sp.1 by the morpholog-ical characteristics of the cultures on malt extract agarplates and by the DNA sequences of the rDNA ITSregion (Tateno pers. comm.). The isolates, that havecultural characteristics similar to X. carpophila, wereinfrequently occurred (<3 %), but were not recordedas cupule interior colonizers when sporocarps ofX. carpophila were observed on cupules (data notshown). D. longistipitata were nor isolated from thecupules. Although life strategies of these fungi are notclear, it is suggested that X. carpophila is not an endo-phyte because this fungus had not been isolated fromlive beech tissues (Tateno pers. comm.).

The remaining mass of soluble carbohydrate in-creased during the decay process. Soluble carbohydratessuch as glucose could be produced during the decayprocess of the holocellulose polymer (Eaton and

Table 2 Changes in contents (mg/g) of organic chemical compo-nents and lignocellulose index (LCI) during decomposition

Months AUR Holocellulose Soluble carbohydrates LCI

0 252 692 6.5 0.733 264 648 6.5 0.716 268 636 7.4 0.7012 287 594 7.7 0.6715 288 565 7.9 0.6618 299 547 9.5 0.6524 323 507 10.9 0.6127 313 505 10.9 0.6230 320 487 12.4 0.60

AUR acid-unhydrolyzable residue

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Hale 1993). However, soluble carbohydrates generallydecreased quickly in the early stages of decompositionduring the decay processes of leaf litter (Osono andTakeda 2005) and woody debris (Ganjegunte et al. 2004)mainly due to leaching and utilization by sugar fungi.Why the net weight of soluble carbohydrate increased incupule litter is not clear. One possible mechanism is thatenclosure of the cupule tissue by hyphae of Xylaria sp.1prevented leaching and invasion of secondary sugarfungi. Species in Xylariaceae form a recalcitrant mel-anized hyphal mat called a ‘‘pseudosclerotial plate’’around their colonies within decay substrates (Lopez-Real and Swift 1975) and maintain these exclusive col-onies until late stages of decomposition (Chapela et al.1988). This plate is very dense and prevents naturalmovement of water (Boddy et al. 1989).

Comparison with leaf and woody litters

When compared with beech leaves (Osono and Takeda2001) and wood (Fukasawa et al. 2009a), beech cupulesare woody and their lignocellulose composition is similarto that of beech wood rather than leaves. However, thedecay process of cupule litter, in which selective holo-cellulose decomposition primarily occurred, was similarto that of beech leaf litter (Osono and Takeda 2001)rather than CWD (Fukasawa et al. 2009a). The majordecomposer of holocellulose in cupule litter, Xylariaspp., was also the same as that in leaf litter (Osono andTakeda 2001). After the selective holocellulose decayprocess, AUR was decomposed in leaf litter by basid-iomycete fungi that replaced Xylaria spp. (Osono andTakeda 2001). In contrast, during the decay process ofbeech CWD, AUR and holocellulose are decomposedsimultaneously by basidiomycetes in the early stages ofdecomposition, and then holocellulose is decomposedselectively in the late stages (Fukasawa et al. 2009a).Among the dominant fungal genera in beech cupulelitter recorded in the present study (Xylaria, Phomopsis,Ascochyta, Nigrospora, and Geniculosporium), onlyPhomopsis was also found in beech CWD (Fukasawaet al. 2009b). Therefore, differences in the fungal com-munity structure between cupule litter and CWD maycontribute to such differences in the decay process oforganic chemical components.

What factors determine the fungal community aftertree tissues die? Possible factors include the size andlocation of plant tissues within the whole tree architec-ture, and contents of secondary metabolites (such aspolyphenols) of the tissues, as well as the life cyclesof fungal decomposers (Rayner and Boddy 1988).Xylariaceous fungi are endophytes of live beech stems(Chapela 1989) as well as leaves (Osono and Mori 2003),twigs (Sahashi et al. 1999) and cupules (Tateno pers.comm.), and latently inhabit the cambium layer(Chapela 1989). Since these fungi start growing imme-diately after tree tissues die, triggered by a decrease ofthe water content of the tissue (Boddy and Griffith

1989), Xylariaceous fungi have an advantage regardingtheir ability to occupy tree tissues compared to otherfungi that must colonize from the outside via airborne orsoil-borne spores or hyphae. This is probably the reasonwhy small leaves and phyllome tissues such as cupules(in the present study) and leaf litters (Osono and Takeda2001) are dominated by endophytic Xylariaceous speciesin their early stages of decomposition. In contrast, stemshave a large proportion of dead tissues, and their poly-phenol content is lower than that of leaves and twigs(Saayman and Roux 1965). These properties of stemsallow airborne or soil-borne basidiomycetes to colonizewood tissues through bark wounds or cut surfaces(Fukasawa et al. 2010). Thus, there may be littleadvantage for endophytes growing from cambium tooccupy wood tissues. Fukasawa et al. (2002) found thatdecay columns of xylariaceous fungi were restricted tothe outer part of beech CWD.

Conclusions

The present study clearly demonstrated that selectivedecomposition of holocellulose occurred initially in thedecay process of Japanese beech cupules. This resembledthe decay process of beech leaf litter rather than beechCWD, although the organic chemical composition ofcupules was more similar to that of wood than to that ofleaves. Xylaria spp., known as holocellulose decompos-ers of beech leaf litter, were also responsible for theholocellulose decomposition of beech cupule litter.Holocellulose selective decomposition stage, in general,is responsible for nitrogen immobilization in leaf litterdecomposition process (Osono and Takeda 2004, 2005)because nitrogen chemically combined to accumulatingAUR components (Berg 1988). Although nutrient con-tents of cupules were not measured in this study, such apulse of cupule litter (masting) and their decompositionmay contributes temporal dynamics of nitrogen immo-bilization in beech forests. However, the complete decayprocess of cupule litter was not revealed in the presentstudy because 77 % of the original mass remained at theend of the 30-month period. Longer-term research willbe needed to reveal later stages of the decay process ofbeech cupule litter.

Acknowledgments We thank members of the Laboratory of ForestEcology and Ashiu Experimental Forest of Kyoto University forhelp with fieldwork; and Dr. E. Nakajima for critical reading of themanuscript. Thanks are extended to a handling editor and twoanonymous reviewers for valuable comments on original manu-script. This study was supported by Global COE Program A06 toKyoto University.

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