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Abstract: Fungal assemblages in live, newly shed and partly decomposed leaves of Camellia japonica were investigated with a clone library analysis to assess ...

Mycologia, 105(4), 2013, pp. 837–843. DOI: 10.3852/12-385 # 2013 by The Mycological Society of America, Lawrence, KS 66044-8897

Assessment of the fungal diversity and succession of ligninolytic endophytes in Camellia japonica leaves using clone library analysis Dai Hirose

ecological roles as endophytes of live leaves (Saikkonen 2007, Sieber 2007) and decomposers of fallen leaves (Osono 2007). The species compositions of fungal assemblages change as live leaves mature, senesce and die and as fallen leaves decompose, in the process of fungal succession (Hudson 1968). Studies have examined the fungal succession in tree leaves with culture-dependent methods, which are believed to underestimate the species richness of fungi because of the selectivity of culture methods and the presence of uncultivable fungi. However, recent advances in culture-independent molecular methods, such as denaturing gradient gel electrophoresis and clone libraries, potentially overcome these problems. Clone library analyses have been used to study fungal assemblages associated with live leaves (Arnold et al. 2007), moss (Kauserud et al. 2008), soils (Landeweert et al. 2003, O’Brien et al. 2005, Taylor et al. 2010), plant roots (Vandenkoornhuyse et al. 2002) and dead leaves (Seena et al. 2008, Poll et al. 2010). However, few studies have examined the fungal successions in live to dead leaves and the life cycles of fungal populations that play central roles in the decomposition of dead leaves, such as in lignin decomposition. The purpose of the present study was to investigate the fungal assemblages in live, newly shed and partly decomposed leaves of Camellia japonica with a clone library analysis to determine the fungal diversity and succession. C. japonica leaves were used because a suite of studies based on a culture-dependent isolation method has examined the fungal colonization and decomposition of these leaves. These include studies of endophytic fungi in live leaves (Osono 2008), the colonization of leaf litter by endophytic fungi (Koide et al. 2005a), fungal succession during decomposition (Koide et al. 2005b) and the potential capacities of endophytic and litter-inhabiting fungi to decompose the litter (Osono and Hirose 2009). Therefore, we hypothesized that the application of a culture-independent method would give comparative information and provide a more comprehensive picture of fungal succession in tree leaves. Studies also have shown that ligninolytic endophytes in the family Rhytismataceae characterize the early stage of decomposition, leading to the occurrence of bleached portions on the surfaces of decomposing C. japonica leaves (Koide et al. 2005a, b; Osono and Hirose 2009). Therefore,

College of Pharmacy, Nihon University, Funabashi, Chiba 274-8555 Japan

Shunsuke Matsuoka Takashi Osono1 Center for Ecological Research, Kyoto University, Shiga 520-2113 Japan

Abstract: Fungal assemblages in live, newly shed and partly decomposed leaves of Camellia japonica were investigated with a clone library analysis to assess the fungal diversity and succession in a subtropical forest in southern Japan. Partly decomposed leaves were divided into bleached and adjacent nonbleached portions to estimate the fungi functionally associated with lignin decomposition in the bleached portions, with an emphasis on Coccomyces sinensis (Rhytismataceae, Ascomycota). From 144 cloned 28S ribosomal DNA (rDNA) sequences, 48 operational taxonomic units (OTUs) were defined based on a sequence similarity threshold of 98%. Forty-one (85%) of the 48 OTUs belonged to the Ascomycota and seven OTUs (15%) to the Basidiomycota. Twenty-six OTUs (54%) were detected only once (singletons). The number of OTUs and the diversity indices of the fungal assemblages in the different leaves were in this order: live leaves . newly shed leaves . bleached portions . nonbleached portions of partly decomposed leaves. The fungal assemblages were similar in newly shed leaves and the bleached portions of partly decomposed leaves. Ligninolytic fungi of the genera Coccomyces, Lophodermium and Xylaria were frequently detected in the bleached portions. OTU3, identified as Coccomyces sinensis, was detected in live and newly shed leaves and the bleached portions of partly decomposed leaves, suggesting that this fungus latently infects live leaves, persists after leaf fall and takes part in lignin decomposition. Key words: decomposition, endophyte, fungi, leaf litter, lignin, subtropical forest INTRODUCTION Fungi are a major component of the microbial assemblages on tree leaves, and they play unique Submitted 9 Dec 2012; accepted for publication 23 Jan 2013. 1 Corresponding author. E-mail: [email protected]

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in the present study partly decomposed leaves were separated into the bleached and adjacent nonbleached portions to identify fungi that are functionally associated with lignin decomposition in the bleached portions and to test the possibility that ligninolytic fungi are present in live leaves. MATERIALS AND METHODS Study site, sample collection and preparation.—The samples were collected in a subtropical evergreen broadleaf forest in the University Forest, University of the Ryukyus, in northern Okinawa, southwestern Japan (26u99N, 128u59E, approximately 250–330 m). The mean annual temperature was 22 C and the annual precipitation was 2456 mm 1992–1999 (Yona Experimental Forest, University of the Ryukyus). Osono et al. (2008) have described further details of the study site. Camellia japonica is a minor component (24 [0.4%] of 5494 stems, $10 cm diam at breast height in a plot [200 3 200 m] dominated by Castanopsis sieboldii [Enoki 2003]). The peak period of litterfall of evergreen tree species, including Camellia japonica, is Mar–Apr (Alhamd and Hagihara 2004). Camellia japonica leaves were collected Apr 2008. Five healthy-looking green leaves attached to five C. japonica trees, 2–4 m tall, were harvested, one leaf per tree. Five newly shed leaves and five partly decomposed leaves on which bleaching was evident were collected from the forest floor beneath the five trees, with one newly shed and one partly decomposed leaf per tree. The leaves were placed in paper bags, which were put in a bag of ice for 3 h, then kept frozen at 220 C for transportation to the laboratory. All samples were stored at 280 C until analysis. The newly shed leaves were easily recognized because the sampling was carried out at peak litter fall. The live and newly shed leaves were cut into small pieces with sterile scissors, avoiding the primary veins and petioles. The partly decomposed leaves were separated into the bleached and nonbleached portions, cut into small pieces, avoiding the primary veins and petioles. We obtained only nonbleached leaf material from three partly decomposed leaves because the surfaces of the other two partly decomposed leaves were entirely bleached. Consequently 18 total leaf samples of C. japonica were used for DNA extraction and clone library analysis. Leaf pieces were washed three or four times with 10 mL sterile 0.005% Aerosol OT solution (w/v) for 1 min and rinsed three times with sterile water in a sterile test tube using a vertical type automatic mixer (S-100, Taitec, Japan). The rinsed pieces were submerged 1 min in 70% ethanol (v/v), surface-sterilized 1 min in a solution of 15% hydrogen peroxide (v/v) and submerged again 1 min in fresh 70% ethanol. The pieces were rinsed three times with sterile water, transferred to sterile filter paper in 9 cm Petri dishes and dried 24 h at 60 C. Clone library analysis.—DNA was extracted from leaf tissues with the DNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s instructions. Leaf tissues were ground with a mortar and pestle in liquid nitrogen, and 20 mg subsamples

of these tissues were placed in tubes for DNA extraction. All extracts were purified with Sephaglas FP and wash buffer (Amersham, USA). Polymerase chain reactions (PCRs) were performed with the HotStarTaq Plus Master Mix Kit (QIAGEN). Each PCR contained 50 mL PCR, 18.5 mL distilled water, 25 mL Master Mix, 3 mL bovine serum albumin (20 mg/mL), 3 mL template DNA and 0.5 mL of each primer (final, 0.25 mM each). The primer pair LR0R (ACCCGCTGAACTTAAGC; Vilgalys unpubl)/LR3 (Vilgalys and Hester 1990) was used to amplify the D1–D2 domain of the 28S rDNA. Each DNA fragment was amplified on a PCR thermal cycler (DNA Engine, Bio-Rad, USA) with this thermal-cycling schedule: initial denaturation at 94 C for 5 min followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 54 C for 30 s and extension at 72 C for 1 min and a final extension at 72 C for 10 min. The reaction mixture was cooled at 4 C for 5 min. PCR products were purified with the QiAquick PCR Purification Kit (QIAGEN, Germany) according to the manufacturer’s instructions. These products were cloned into the pCR4-TOPO vector with the TOPO TA cloning kit (Invitrogen) according to the manufacturer’s instructions. Recombinant One Shot DH5aT1 Escherichia coli colonies were randomly picked and screened directly for inserts with colony PCR using primers specific for the vector. The PCR products from the positive clones were purified in the manner described above and sequenced by FASMAC Co. (Kanagawa, Japan). Eight colonies were sequenced from each leaf sample. The sequencing reactions were performed in a GeneAmp PCR System 9700 (Applied Biosystems, USA) with a BigDye Terminator 3.1 (Applied Biosystems) according to the protocols supplied by the manufacturer. The fluorescently labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The samples were resuspended in formamide and subjected to electrophoresis in an ABI 3730xl sequencer (Applied Biosystems). The sequences determined in the present study were deposited in the DNA Data Bank of Japan (DDBJ accession numbers AB618821–AB618964; SUPPLEMENTARY TABLE I). Sequences were clustered into operational taxonomic units (OTUs) with the Claident software package (http:// www.fifthdimension.jp/products/claident/), with a 98% similarity threshold. The sequences of each OTU were compared with the available rDNA sequences in the GenBank database with BLASTN queries (Altschul et al. 1990) and taxonomically assigned based on the best BLAST match to a known organism. Data analysis.—An OTU was considered major when it was represented by more than three of the eight clones isolated from each leaf sample of the four leaf types. The relative frequencies of the individual OTUs on each of the four leaf types were calculated according to this equation (Pianka 1973): relative frequency (%) 5 number of clones with the OTU/total number of clones 3 100. The total number of clones was 40 for the live leaves, the newly shed leaves and the bleached portions of the partly decomposed leaves and 24 for the nonbleached portions of the partly decomposed leaves.

HIROSE ET AL.: FUNGAL SUCCESSION IN CAMELLIA LEAVES The total number of OTUs in each leaf type was considered to indicate OTU richness (S). Simpson’s diversity index (D) and equitability (E) were calculated with these equations: D 5 1/g Pi 2, E 5 D/S, where Pi is the ratio of the number of clones of the ith OTU to the total number of clones for each leaf type. D indicates the character of an assemblage, taking into account both the relative frequency pattern and the OTU richness. E indicates the evenness with which the OTUs are distributed among the clones. Analysis of variance (ANOVA) was used to test the differences in the mean numbers of OTUs in the four leaf types. JMP 6.0 for Macintosh was used for the analysis. Rarefaction curves were generated to depict the effect of the number of clones examined on the number of fungal OTUs for the four leaf types using EstimateS 8.2 (Colwell 2006). To compare the fungal assemblages of the four leaf types, we used nonmetric multidimensional scaling (NMDS) with the Bray-Curtis distance metric. This analysis was carried out in the R environment (http://www. r-project.org) with the vegan package (Oksanen et al. 2008).

RESULTS Fungal OTUs.—A total of 48 OTUs were obtained from Camellia japonica leaves (TABLE I), 41 of which (85%) belonged to the Ascomycota and seven (15%) to the Basidiomycota. Twenty-six OTUs (54%) were detected only once (singletons) and 15, five, four and two of these OTUs occurred in live leaves, newly shed leaves and the bleached and nonbleached portions of partly decomposed leaves respectively (TABLE I). Nine OTUs were regarded as major (TABLE I), all belonging to the Ascomycota. OTU2, OTU3 and OTU7 were 100% identical to the sequences of Lophodermium sp., Coccomyces sinensis and Xylaria sp. respectively, determined from ascocarps on fallen leaves of C. japonica at the study site (Osono 2009). OTU11, detected in newly shed leaves with a frequency of occurrence of 7.5%, had 100% sequence identity with an unidentified species in the Dermateaceae isolated from C. japonica leaves (Osono and Hirose 2009). No fungal OTU was detected in all four types of leaves. Fungal assemblages.—The total and mean numbers of OTUs, the diversity indices and the equitability were in this order: live leaves . newly shed leaves . bleached portions of partly decomposed leaves . nonbleached portions of partly decomposed leaves (TABLE II, FIG. 1). The mean number of OTUs was significantly greater in live leaves than in the nonbleached portions of partly decomposed leaves (ANOVA, P , 0.05). NMDS ordination showed changes in the fungal assemblages from live to newly shed leaves to partly decomposed leaves but similar assemblages in the newly shed leaves and the bleached portions of partly decomposed leaves (FIG. 2).

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Succession of major OTUs.—The changes in relative frequencies of the nine major OTUs (FIG. 3) show that OTU4 (most closely related to Pseudophloeospora eucalypti) and OTU9 (most closely related to Mycoleptodiscus terrestris) were found most frequently in live leaves but accounted for only 20% of the total clones. The other 24 OTUs occurred in live leaves but with low relative frequencies (2.5%–7.5%). OTU9 was not detected in newly shed leaves, whereas the relative frequencies of OTU3 (Coccomyces sinensis) and OTU4, which were detected in the live leaves, were increased in the newly shed leaves. OTU1 (most closely related to Hyalodendriella betulae), OTU2 (Lophodermium sp.) and OTU6 (most closely related to Falcocldium thailandicum) also were found frequently in newly shed leaves. These five major OTUs accounted for 65% of the total clones in the newly shed leaves. The compositions of the major OTUs differed markedly between the bleached and adjacent nonbleached portions of the partly decomposed leaves. OTU2, OTU3, OTU5 (most closely related to P. eucalypti) and OTU7 (Xylaria sp.) were the major components of the bleached portions, accounting for 77.5% of the total clones, whereas in the nonbleached portions of the partly decomposed leaves, OTU1 and OTU8 (most closely related to Dactylaria parvispora) were the dominant OTUs, accounting for 75% of the total clones. DISCUSSION A total of 144 clones were clustered into 48 OTUs (with a similarity threshold of 98%; TABLE I). The fact that the Ascomycota and Basidiomycota were the major components of the clone libraries from Camellia japonica leaves is consistent with the results of previous studies, but the relative proportions were more biased toward the Ascomycota (85% of the total number of OTUs) compared to the Basidiomycota (15%) than in previous studies of plant substrates (Vandenkoornhuyse et al. 2002, O’Brien et al. 2005, Kauserud et al. 2008, Poll et al. 2010), which reported proportions of 35–63% for Ascomycota and 27–41% for Basidiomycota. Previous culture-dependent studies of the endophytic fungi associated with C. japonica leaves reported the frequent isolation of species of Geniculosporium and Colletotrichum from live leaves (Koide et al. 2005a, Osono 2008) and Khuskia, Eupenicillium and Pestalotiopsis from fallen leaves (Koide et al. 2005b). These fungal genera were not detected in the present study, which can be partly attributed to differences in the study sites, which are 1150 km apart, and in the selectivity of the isolation and molecular methods used. In contrast, Lophodermium sp. (OTU2), Coccomyces sinensis (OTU3),

840 TABLE I.

MYCOLOGIA Relative frequencies (%) and BLAST query results for 48 OTUs from the leaves of Camellia japonica Frequency

BLAST

OTU

LLa

NSb

BLc

NBd

OTU1*e OTU2* OTU3* OTU4* OTU5* OTU6* OTU7* OTU8* OTU9* OTU10 OTU11 OTU12 OTU13 OTU14 OTU15 OTU16 OTU17 OTU18 OTU19 OTU20 OTU21 OTU22 OTU23 OTU24 OTU25 OTU26 OTU27 OTU28 OTU29 OTU30 OTU31 OTU32 OTU33 OTU34 OTU35 OTU36 OTU37 OTU38 OTU39 OTU40 OTU41 OTU42 OTU43 OTU44 OTU45 OTU46 OTU47 OTU48

0.0 0.0 2.5 10.0 0.0 0.0 0.0 0.0 10.0 0.0 0.0 7.5 0.0 0.0 5.0 0.0 5.0 5.0 2.5 5.0 5.0 5.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

7.5 15.0 12.5 15.0 2.5 15.0 0.0 0.0 0.0 0.0 7.5 0.0 7.5 0.0 0.0 2.5 0.0 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 2.5 2.5 2.5 2.5 0.0 0.0 0.0 0.0 0.0 0.0

0.0 27.5 22.5 0.0 15.0 0.0 12.5 0.0 0.0 7.5 0.0 0.0 0.0 2.5 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 2.5 2.5 2.5 0.0 0.0

58.3 0.0 0.0 0.0 8.3 0.0 0.0 16.7 0.0 4.2 0.0 0.0 0.0 4.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.2 4.2

a

Closest match in GenBank (accession no.) Hyalodendriella betulae (EU040232) Lophodermium sp. (AB512357) Coccomyces sp. (AB512350) Pseudophloeospora eucalypti (HQ599593) Pseudophloeospora eucalypti (HQ599593) Falcocladium thailandicum (EU040216) Xylaria sp. (AB512361) Dactylaria parvispora (EU107296) Mycoleptodiscus terrestris (JN711859) Rhodotorula yarrowii (AF189971) Dermateaceae sp. (AB298438) Trichothecium roseum (EU552162) Bisporella citrina (EU940087) Coccomyces sp. (AB512350) Derxomyces mrakii (JN939797) Lophodermium sp. (AB512353) Lophiotrema nucula (GU301837) Cyphellophora hylomeconis (EU035415) Cryptodiaporthe vepris (EU683070) Camarographium koreanum (JQ044451) Readeriella guyanensis (FJ493211) Cladophialophora bantiana (AB363799) Monacrosporium tentaculatum (AY902792) Otthia spiraeae (GU205226) Candida pseudointermedia (FJ986609) Cochliobolus nodulosus (JN600997) Byssoloma leucoblepharum (AY756317) Derxomyces simaoensis (JN939786) Opegrapha celtidicola (EU704094) Valsaria ceratoniae (EU040213) Cryptococcus sp. (EU678944) Enterographa hutchinsiae (EU704089) Brycekendrickomyces acaciae (FJ839641) Ceramothyrium carniolicum (FJ358232) Mycoleptodiscus terrestris (JN711859) Elsinoe verbenae (JN940391) Cyphellophora eucalypti (GQ303305) Leotiomycetes sp. (JQ759938) Rhodotorula sp. (AY731807) Kriegeria eriophori (AY745728) Cryptodiaporthe vepris (EU683070) Lophodermium sp. (AB512357) Coccomyces sp. (AB512350) Uncultured Hymenoscyphus (AY394699) Chalara hughesii (FJ176250) Sistotrema coroniferum (AM259215) Uncultured Coniochaeta (JF449668) Dactylaria parvispora (EU107296)

LL, live leaves. NS, newly shed leaves. c BL, bleached portions of partly decomposed leaves. d NB, nonbleached portions of partly decomposed leaves. e Asterisks indicate major OTUs (see text). b

Query coverage

Maximum identity

100% 98% 96% 99% 99% 100% 98% 100% 99% 99% 98% 99% 99% 98% 99% 98% 99% 99% 97% 99% 99% 99% 99% 99% 100% 99% 99% 99% 99% 99% 99% 98% 99% 99% 99% 99% 99% 98% 99% 99% 100% 99% 99% 100% 100% 99% 99% 100%

94% 100% 100% 97% 96% 98% 100% 94% 95% 99% 100% 97% 94% 92% 98% 98% 99% 97% 96% 96% 98% 93% 90% 99% 99% 97% 96% 97% 84% 90% 99% 93% 99% 97% 92% 93% 97% 95% 99% 94% 94% 93% 97% 98% 99% 94% 91% 92%

HIROSE ET AL.: FUNGAL SUCCESSION IN CAMELLIA LEAVES TABLE II.

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Total and mean numbers of OTUs, diversity and equitability of fungal assemblages in leaves of Camellia japonica Partly decomposed leaves

Total number of leaves Total number of clones Total number of OTUs Number of singleton OTUs (% total number of OTUs) Mean number of OTUsa Simpson’s diversity index Equitability

Live leaves

Newly shed leaves

Bleached portions

Nonbleached portions

5 40 26 15 (58%) 6.2 6 0.7 a 19.5 0.75

5 40 15 5 (33%) 3.8 6 0.5 ab 9.5 0.63

5 40 11 4 (36%) 3.8 6 0.6 ab 5.8 0.52

3 24 7 2 (29%) 2.7 6 0.9 b 2.6 0.37

a

Values indicate means 6 standard errors. Numbers with the same letters are not significantly different at the 5% level by Tukey’s HSD test.

Xylaria sp. (OTU7) and an unidentified species of Dermateaceae (OTU11) detected in the clone libraries in the present study previously were isolated from C. japonica leaves (Koide et al. 2005a, b) and/or their ascocarps were observed on the leaf surfaces (Osono 2009). This demonstrates the successful molecular confirmation of these fungal species in leaf tissues. The greater total and mean numbers of OTUs in the live leaves compared to the newly shed or the partly decomposed leaves (TABLE II) indicate greater fungal OTU richness within the live leaves used for the present study. The latent colonization of live leaves by endophytic fungi is known to be highly localized (Suske and Acker 1987, Arnold 2008), even to within single epidermal cells (Stone 1987). The colonization of live leaves by yeasts (such as in the case of OTU15, OTU25, OTU28, OTU31) also must

FIG. 1. Rarefaction curves depicting the effects of the number of clones examined on the number of fungal OTUs in live (%) and newly shed leaves (#) and in bleached (n) and nonbleached portions (e) of partly decomposed leaves of Camellia japonica.

be localized because they have a single-cell habit and contribute to the higher fungal richness in live leaves. In contrast, individual fungal colonies in newly shed or partly decomposed leaves are considered to be generally larger because of the saprobic colonization of leaf tissues by the mycelia of decomposer fungi. This is exemplified in partly decomposed leaves with visible bleached portions delimitated by black zone lines, each of which corresponds to the colony of a single fungal mycelium (Hirose and Osono 2006). The lower fungal diversity in the nonbleached portions of the partly decomposed leaves is also partly attributable to the smaller number of samples used in the present study.

FIG. 2. Multivariate ordination of fungal assemblages in Camellia japonica leaves, using nonmetric multidimensional scaling (NMDS) with Bray-Curtis dissimilarity coefficients. Symbols identical to FIG. 1.

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FIG. 3. Relative frequencies (%) of major fungal OTUs in Camellia japonica leaves. The numbers in parentheses indicate the numbers of OTUs other than the major ones, which had lower relative frequencies. Asterisks indicate fungal OTUs with ligninolytic activity: OTU2, Lophodermium sp.; OTU3, Coccomyces sinensis; and OTU7, Xylaria sp. (see text).

The differences in the fungal assemblages in the four leaf types (FIG. 2) reflect the fungal succession that occurred as the leaves fell and decomposed. Some endophytic fungi present in the live leaves disappeared after leaf death (e.g. OTU9), whereas others increased in frequency in the newly shed leaves (e.g. OTU3, OTU4) (TABLE I), consistent with reports summarized by Osono (2006). The fungal assemblage in the newly shed leaves was similar to that in the bleached portions of the partly decomposed leaves (as shown in NMDS, FIG. 2), which demonstrates that the persistence of the fungi associated with newly shed leaves (i.e. C. sinensis, Lophodermium sp.) led to the bleaching of the leaf litter. Koide et al. (2005a) have already shown that C. sinensis (mistakenly denoted as C. nipponicum) bleaches the C. japonica leaf litter soon after the leaves die. Pure culture tests showed that species of Coccomyces, Lophodermium and Xylaria collected at the study site can remove lignin and bleach leaf tissues (Osono 2009). In contrast, the replacement of these fungi with OTU1 and OTU8 may result in the nonbleached leaf tissues, in which cellulose is selectively removed (Koide et al. 2005b). Thus, the results of the present study are consistent with those of previous culture-dependent studies and suggest that fungal colonization and succession have significant effects on the patterns of relative decomposition of the organic chemical components of C. japonica leaves. Koide et al. (2005a) isolated Coccomyces sinensis from both newly shed and decomposing leaves of C. japonica. In their study, when newly shed leaves that had been sterilized to exclude previously established

endophytes were incubated on the forest floor, there was no occurrence of the bleached portions caused by this fungus. This suggests that this species was already present endophytically in the live leaves. The molecular method used in the present study successfully detected C. sinensis in both symptomless live and newly shed leaves (TABLE I, FIG. 3), providing further evidence that the fungus infects live leaves asymptomatically. Coccomyces sinensis thus appears to have a life cycle similar to that reported for Rhabdocline parkeri, a hemiphacidiaceous endophyte latent in healthy-looking needles of Pseudotsuga menziesii (Stone 1987). Deckert et al. (2001) documented the latent colonization of live leaf tissue by Lophodermium sp., which have a similar life cycle (Osono and Hirose 2011), but in the present study it was not detected in live leaves, possibly because of the low infection rate in live leaves and the small number of live leaves examined. Because the ligninolytic activity of endophytic fungi is unique in terms of its ecological and evolutionary significance (Osono 2006), further studies are required to examine the diversity, mode of infection and phylogeography of these rhytismataceous endophytes. ACKNOWLEDGMENTS We thank Dr A. Takashima and the staff of the Yona Experimental Forest, University of the Ryukyus for their help with the fieldwork and Ms. C. Sakaguchi for her help with the data analyses. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (No. 23770083), the Global COE Program A06 to Kyoto University and the Academic Frontier Project for Private Universities, a matching fund subsidy from MEXT.

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