f u n g a l b i o l o g y 1 1 7 ( 2 0 1 3 ) 1 9 1 e2 0 1 journal homepage: www.elsevier.com/locate/funbio A comparison of fungal endophytic community diversity in tree leaves of rural and urban temperate forests of Kanto district, eastern Japan Emi MATSUMURA*, Kenji FUKUDA Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwa-no-ha, Kashiwa, Chiba 277-8563, Japan article info abstract Article history: To clarify the effects of forest fragmentation and a change in tree species composition fol- Received 16 August 2012 lowing urbanization on endophytic fungal communities, we isolated fungal endophytes Received in revised form from the foliage of nine tree species in suburban (Kashiwa City, Chiba) and rural (Mt. Waga- 16 December 2012 kuni, Ibaraki; Mt. Takao, Tokyo) forests and compared the fungal communities between sites Accepted 15 January 2013 and host tree species. Host specificity was evaluated using the index of host specificity (Si), Available online 4 February 2013 and the number of isolated species, total isolation frequency, and the diversity index were Corresponding Editor: calculated. From just one to several host-specific species were recognized in all host tree spe- Martin I. Bidartondo cies at all sites. The total isolation frequency of all fungal species on Quercus myrsinaefolia, Quercus serrata, and Chamaecyparis obtusa and the total isolation frequency of host-specific Keywords: species on Q. myrsinaefolia, Q. serrata, and Eurya japonica were significantly lower in Kashiwa b diversity than in the rural forests. The similarity indices (nonmetric multidimensional scaling (NMS) Endophytic fungal community and CMH) of endophytic communities among different tree species were higher in Kashiwa, Forest isolation as many tree species shared the same fungal species in the suburban forest. Endophytic Host specificity fungi with a broad host range were grouped into four clusters suggesting their preference Urbanization for conifer/broadleaves and evergreen/deciduous trees. Forest fragmentation and isolation by urbanization have been shown to cause the decline of host-specific fungal species and a decrease in b diversity of endophytic communities, i.e., endophytic communities associated with tree leaves in suburban forests were found to be depauperate. Crown Copyright ª 2013 Published by Elsevier Ltd on behalf of The British Mycological Society. All rights reserved. Introduction Forest types are largely determined by climate, namely temperature and precipitation (Whittaker 1970). It is well known that the diversity of terrestrial plants is highest in the tropical rain forests, whereas in temperate regions, a few tree species dominate the forest canopy. Many fungi, such as decomposers, wood-decaying polypores, endophytes in the rhizosphere and phyllosphere, ectomycorrhizal (ECM) fungi, etc., depend upon forest plants. Fungal diversity per single host in tropical forests is known to be higher than that in temperate forests; however, species diversity per forest is not higher in the tropics for polypores (Drechsler-Santos et al. dhiou et al. 2010), and arbuscular mycor2010), ECM fungi (Die rhizal fungi (AMF) (Zhao et al. 2003). This is because most fungal groups show higher host specificity in temperate forests than in tropical forests (Hattori 2005), and this is particularly true for ECM fungi (Tedersoo & Nara 2010). Thus, ECM fungi * Corresponding author. Tel./fax: þ81 4 7136 4750. E-mail address: [email protected] (E. Matsumura). 1878-6146/$ e see front matter Crown Copyright ª 2013 Published by Elsevier Ltd on behalf of The British Mycological Society. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.01.007 192 display hyperdiversity in mixed conifer-broadleaf forest in temperate regions, which is a consequence of host and fungal diversity supporting each other (Ishida et al. 2007). Endophytic fungi that asymptomatically infect healthy plant tissues are known to be ubiquitous in every organ of every plant species (Petrini 1986). Most tree-leaf endophytes are decomposers or weak pathogens, which latently infect the leaves with horizontal infection by spores rather than systemic infection (Wilson 1996; Kaneko & Kaneko 2004). Thus, abiotic factors, such as temperature, rainfall, and humidity, and biotic factors, such as host density, may strongly affect the occurrence of endophytic fungi (Carroll 1995; Schulz & Boyle 2005; Sieber 2007). Under natural conditions, the tree-leaf endophyte communities of tropical forests tend to be dominated by fungal species that have a broad host range (Suryanarayanan et al. 2002; Schulz & Boyle 2005). In temperate forests, the endophytic communities in host species of the same plant family tend to be dominated by closely-related endophyte species (Petrini & Carroll 1981; Sieber et al. 1991; Sahashi et al. 2000; Ragazzi et al. 2003; Helander et al. 2007; Hashizume et al. 2008). There are clear differences in endophytic fungal communities in temperate forests between gymnosperms and angiosperms, which are dominated by Helotiales and Diaporthales, respectively. These two orders are estimated to have diverged 300 Ma ago, suggesting that the dominant endophytes have coevolved with their hosts (Sieber 2007). The number of species on an island is determined by a dynamic balance between immigration and extinction rates of species, which depend on the size of the island and the distance from a source of propagules (MacArthur & Wilson 1967); this pattern is true for endophytic fungi in birch leaves growing on islands (Helander et al. 2007). This theory can also explain differences in endophyte community characteristics between tropical and temperate forests. Namely, in tropical forests, where tree species are diverse and individuals of the same species are scattered, host trees can be considered isolated islands for host-specific endophytic fungi, while the forest is a continuous mainland for fungi with a broad host range. In contrast, in temperate and boreal forests that contain a few dominant tree species, hostspecific fungi can dominate the endophyte community. Decreases in fungal abundance or species diversity in urban forests have been reported for many fungal groups (Ochimaru & Fukuda 2007; Newbound et al. 2010; Bainard et al. 2011), including leaf endophytes (Jumpponen & Jones 2010), but these reductions have only been attributed to direct factors such as pollution and heat-islands. Habitat disjunction and fragmentation, however, also influence not only the flora and fauna (Iida & Nakashizuka 1995; Sewell & Catterall 1998), but also the mycoflora (Drinnan 2005). We hypothesize that an isolated environment will more strongly affect host-specific fungal species according to the theory of island biogeography (MacArthur & Wilson 1967). Specifically, endophytic fungi with narrow host ranges will be more severely affected by limited dispersal than fungi with a broad host range. Most temperate forests in Japan are mixed and codominated by tree species of diverse phylogeny (gymnosperms and angiosperms) and different life forms (evergreen and deciduous). East Asian forests, including Japanese forests, are more diverse than temperate forests in Europe and North America. As a result, fungi with a broad host range might be E. Matsumura, K. Fukuda more frequently isolated in temperate forests in Japan than in Europe and North America (Osono 2008; Hashizume et al. 2008). Therefore, Japanese temperate forests provide a good model for examining the effect of fragmentation on both host-specific fungi and species with a broad host range. Therefore, the aim of this study was to reveal the difference between suburban and natural forests with respect to fungal endophytic community diversity in temperate regions. We isolated leaf endophytic fungi from nine tree species including five common species in three sites, a suburban forest and two rural forests. Materials and methods Materials and study sites All tree leaves were sampled during the summer in 2006 and 2007 in two rural forests and a suburban forest (Mt. Wagakuni: July 19, August 20, and September 21, 2007; Mt. Takao: August 20 and September 29, 2006, and July 26, 2007; Kashiwa: July 30 and September 7, 2006, and September 30, 2007; Table 1). In total, nine species from seven genera in four families, including five tree species that were common to all sites, were surveyed to determine endophytic communities (Table 1). All climatic data were obtained from the nearest automated meteorological data-acquisition system (AMeDAS) weather station. The Mt. Wagakuni (WG) site has a cool-temperate climate. The upper part of the site was secondary forest dominated by Carpinus spp., with some deciduous broadleaved trees. The lower part comprises plantations of Japanese cedar [Cryptomeria japonica D. Don] and Hinoki cypress [Chamaecyparis obtusa (Sieb. et Zucc.) Endl.], with Quercus myrsinaefolia Bl., Eurya japonica Thunb., and Aucuba japonica Thunb. mixed in the subtree layer. A climax stand of beech (Fagus crenata Blume) was located next to the study site (Fig 1). Four Quercus species (two evergreen species of subgenus Cyclobalanopsis, Q. myrsinaefolia and Quercus acuta Thunb., and two deciduous species of subgenus Quercus (or Lepidobalanus), Quercus serrata and Quercus crispula Bl.) occurred at this site. The Mt. Takao (TK) site is characterized by both cool- and warm-temperate climate. Evergreen conifers (Abies firma Sieb. et Zucc. and Tsuga sieboldii Carr.) and evergreen broadleaved Quercus spp. dominated the stands, either alone or in combination, with some stands of Fagus japonica Maxim. Plantations of Japanese cedar and Hinoki cypress were located next to the study area. In total, ten Quercus species (six evergreen species, Q. myrsinaefolia, Quercus glauca Thunb., Q. acuta, Quercus salicina Bl., Quercus sessilifolia Bl., Quercustakaoyamensis Makino, and three deciduous species, Q. serrata, Q. crispula, and Quercusanguselepidola Nakai) occurred at this site (Yoshiyama 1985). The Kashiwa (KS) site has a warm-temperate climate and is an isolated 1.5-ha suburban forest (Fig 1). This stand is a secondary forest, dominated by evergreen and deciduous Quercus spp. The succession of this forest will progress to climax evergreen Q. myrsinaefolia, Castanopsis sieboldii (Makino) Hatus. ex T. Yamaz. et Mashiba, and Machilus japonica Sieb. et Zucc. ex Bl. (e.g., Numata 1969). Stands were inferred to be approximately 60 y old (Yamashita et al. 2007). In this stand, some planted conifer trees (C. japonica and C. obtusa) were also found. In total, ten Quercus species (one evergreen, Fungal endophytic diversity in urban forest 193 Table 1 e Site conditions and host tree species. Site Mt. Wagakuni Land use Location Elevation (m asl) Mt. Takao Kashiwa Rural N36 190 1900 E140 120 0400 400 Rural N35 370 3000 E139 140 3700 400 Urban N35 540 0700 E139 560 0000 20 Climates (annual) Average temperature ( C) Max temperature ( C) Min temperature ( C) Rainfall (mm) 10.8 27 5.8 1330 12.7 28.9 3.4 1571.8 14.4 30.3 0.7 1335 Climates (sampling periods) Average temperature ( C) Max temperature ( C) Min temperature ( C) Average rainfall (mm) 22 32.5 14.3 178 22.4 33.7 15.8 240.3 25 34 17.5 180 Life form Deciduous Host species Quercus serrata Murray Castanea crenata Siebold et Zucc. Fagus japonica Maxim. Carpinus tschonoskii Maxim. Mt. Wagakuni þ Mt. Takao þ Kashiwa þ þ Evergreen Eurya japonica Thunb. var. japonica Quercus myrsinaefolia Blume Quercus glauca Thunb. þ þ þ þ þ þ þ Conifer Chamaecyparis obtusa (Siebold et Zucc.) Endl. Cryptomeria japonica (L.f.) D.Don þ þ þ þ þ þ Q. myrsinaefolia, and two deciduous trees, Q. serrata and Quercus acutissima Carruth.) occurred at this site. We selected three individuals from each tree species, and fifteen mature healthy leaves were collected from each selected individual during each sampling period. Therefore, one sample unit comprised 135 leaf disks or segments (three sampling periods 15 leaves three individuals from each host species at each site). Endophyte isolation Fungal isolation was conducted within 48 h of sample collection. The leaves of collected samples were washed under running tap water for 12 h. Then, they were sterilized in 80 % ethanol for 1 min, 1 % NaOCl for 1 min, 80 % ethanol for an additional 1 min, and then rinsed twice in sterile water. After drying the leaves on sterile paper, two leaf disks (6 mm in diameter) were punched out from broadleaf species, Cryptomeria needles were cut into 1-mm lengths, and a pair of Chamaecyparis scales were cut into 3-mm squares. The samples were incubated, with five segments per dish, on modified half-strength potato dextrose agar (PDA) containing chloramphenicol (M-1/ 2 PDA; 19.5 g PDA (Merck, Darmstadt, Germany), 9.0 g of plate count agar (PCA; Merck), and 600 mg of chloramphenicol/L) in a Petri dish (90-mm diameter) and kept at 20 C under dark conditions. Emerged colonies were transferred into new Petri dishes and incubated under the same conditions at room temperature. The pure cultures were exposed to natural daylight if not sporulated. Sporulated isolates were identified morphologically to genus or species level. Sterile isolates were grouped into morphotypes according to their cultural and morphological characteristics, and more than one isolate from each morphotype was identified by DNA analysis. þ þ Fungal identification by DNA analysis Fungal DNA was extracted from pure culture mycelia using a DNeasy Plant Mini Kit (Qiagen). The rDNA ITS region was amplified using ITS5 and ITS4 primers (White et al. 1990). PCR amplifications were performed in a reaction mixture containing AmpliTaq Gold master mix (Applied Biosystems) or GoTaq master mix (Promega), DNA extracts, and the primer pair. Thermocycler settings were 10 min initial denaturation at 94 C, followed by 30 cycles of 30 s at 94 C, 1 min at 51 C, 1 min at 72 C, with a 10-min final extension at 72 C. The PCR products were purified by Microcon-100 (Milipore) and sequenced using a 3130 Genetic Analyzer (Applied Biosystems) after the sequence reaction with a BigDye Terminator version 3.1 Cycle Sequencing kit. Aligned sequence data were collected from BLAST searches. Isolates with over 97 % sequence similarity were identified as the same species, and those with over 95 % sequence similarity were identified as the same genus. Sequence data were deposited in GenBank (accession numbers are included in Table 5). Data analysis The isolation frequency (IF) of an endophyte taxon was calculated as follows: IF (%) ¼ Ni/Nt 100, where Ni is the total number of leaf disks/segments from which the fungus was isolated, and Nt is the total number of leaf disks/segments. Fungal species with IF <3 % were regarded as rare. This included species of Cladosporium, Fusicoccum, Penicillium, Trichoderma, Biscogniauxia, Nodulisporium, Xylariaceous fungi, yeasts, and unidentified fungi. Host specificity was evaluated for each fungal taxon by the index of host specificity (Si), which indicates both the number 194 E. Matsumura, K. Fukuda Fig 1 e Study site, Satellite image of the Kanto area (left upper, Google Earth) and vegetation maps for each site (1 km square). of host species and the evenness of their abundances as (Rohde 1980) X X Xij =nj ; Si ¼ Xij =nj hij where, for the ith fungal species, xij is the number of leaf segments where the ith species was isolated from the jth host species; nj is the number of leaf segments of the jth species examined; and hij is the rank of host species j based on IF (the species with the greatest IF has rank 1). After that, the distribution of the Si index was fitted to a mixture of normal distributions by JMP ver. 9.03. The diversity of endophytic communities was evaluated by use of the ShannoneWiener H0 and evenness index E5 (also known as the modified Hill’s ratio). The E5 index was used because it is relatively unaffected by the number of species or the sample size (Ludwig & Reynolds 1988). X pi loge pi H0 ¼ l¼ X p2i 0 E5 ¼ ð1=l 1Þ= eH 1 ; where pi is the frequency of occurrence of each species. To compare fungal diversity between sites, five tree species that were common to all sites were used. To compare a diversity, the total number of fungal species in each site was estimated from the species accumulation curve using ESTIMATES ver. 8.2 (Colwell 2006). To compare b diversity, we Order Botryosphaeriales Xylariales Diaporthales Diaporthales Helotiales Capnodiales Diaporthales Pleosporales Capnodiales Diaporthales Diaporthales Diaporthales Xylariales Glomerellales Diaporthales Xylariales Dothideales Botryosphaeriales Glomerellales Pleosporales Trichosphaeriales Xylariales Diaporthales Mycosphaerellales Glomerellales Xylariales Xylariales Fungal species Phyllosticta cryptomeriae White sterile Cs2 Gnomonopsis sp. White sterile PH1 Tubakia sp. 2 Pezicula sp. Pseudocercospora sp. Tubakia sp. 1 Ascochyta fagi Ramichloridium cerophilum Yellow sterile GL2 Discula sp. 1 Yellow sterile GL1 Tubakia sp. 3 Discula sp. 2 Rosellinia sp. 2 White sterile DT1 Glomerella sp. (Colletotrichum sp. 3) White sterile WM1 Phomopsis sp. 2 Xylaria sp. Botryosphaeria dothidea WHS Phyllosticta capitalensis Colletotrichum acutatum Alternaria sp. Nigrospora sp. 2 Astrocystis sp. Phomopsis spp. White sterile WH7 Aureobasidium sp. Colletotrichum gloeosporioides Muscodor fengyangensis Pestalotiopsis sp. Cryptomeria japonica Cj Si WG TK KS 1.00 57.0 74.8 57.0 0.99 0.99 0.99 0.99 0.98 0.98 0.97 0.96 0.94 0.93 0.90 0.86 0.84 0.82 0.78 0.75 0.69 0.67 0.66 0.64 0.64 0.64 0.63 0.63 Chamaecyparis obtusa Co WG TK KS Eurya japonica Ej WG TK Quercus glauca Qg KS TK Quercus myrsinaefolia Qm WG TK KS 7.8 þ þ 7.4 11.9 þ 10.7 þ 14.8 33.0 12.2 19.3 1.5 13.0 7.0 6.7 þ þ þ þ þ þ 43.7 þ þ þ 5.2 9.6 13.7 þ þ þ 11.1 11.1 4.4 þ þ þ 0.63 0.58 0.55 0.55 0.53 0.53 0.50 þ 5.9 6.7 5.9 þ 10.4 þ 0.48 33.3 0.43 þ þ 9.6 14.1 þ þ þ þ 4.4 þ þ 9.6 þ þ 25.2 þ þ 5.2 4.4 5.2 þ þ þ þ TK KS WG þ þ þ 11.5 48.1 þ 8.1 12.6 3.3 þ þ 8.1 þ 10.4 6.7 þ 7.8 4.8 þ þ 5.6 17.0 5.6 5.9 15.2 þ þ þ þ þ 17.0 þ þ 5.2 52.2 þ þ 15.6 47.4 5.6 5.6 þ þ þ þ 4.8 þ þ þ þ 7.8 þ þ þ 8.1 þ þ þ þ 17.0 þ þ þ þ 27.4 þ þ þ þ þ þ 5.6 þ þ 8.1 þ þ þ þ 7.0 15.2 þ þ 8.5 21.5 5.6 þ þ þ þ þ þ 4.1 þ 8.1 5.9 21.1 5.6 7.8 þ þ þ 62.6 þ þ þ 28.9 þ KS þ þ þ TK þ þ þ þ WG Fagus Castanea Carpinus japonica crenata tschonoskii Fj Cc Ct þ þ þ 5.2 þ þ Quercus serrata Qs 58.5 þ 27.0 þ þ 7.8 þ þ þ þ þ 14.8 þ 5.2 8.5 5.9 þ þ þ 43.0 14.4 þ þ þ þ 4.8 þ þ 31.1 6.3 þ 30.4 þ þ þ þ 4.1 þ þ 10.7 þ þ þ 54.8 þ þ þ þ þ 14.8 þ 17.4 þ þ þ 4.4 9.3 þ 4.8 þ þ þ 5.6 þ 11.5 þ 25.9 þ þ þ þ 13.3 5.2 þ þ 6.7 33.7 þ þ þ 9.6 3.3 4.4 þ 34.8 þ þ 11.1 þ þ þ þ þ þ þ 4.8 7.0 þ 6.7 þ 7.8 23.7 þ þ 7.0 þ 20.7 18.5 39.3 12.6 þ 3.7 þ 195 ‘þ’ means IF lower than 3.0 %. Host species Fungal endophytic diversity in urban forest Table 2 e IF (%) and host-specificity index of major endophytic fungi on the leaves of nine tree species in three sites. 196 E. Matsumura, K. Fukuda Table 3 e Comparison of the IF and diversity estimations for endophytes among three sites. Host species site Cryptomeria japonica WG 165 57.0 TK 110 74.8 KS WG TK KS Eurya japonica WG TK Quercus myrsinaefolia KS WG 102.2a 81.5ab 51.9b 110.0 98.5 110 7.4 14.8 9.6 13.3a 6.9ab 5.7b TK KS Quercus serrata WG TK KS 88.5ab 131.1a 75.9b 157.4ab 144.8a 136.7b 10.6b 30.4a 4.9b 62.6a 58.5a 27.0b Sum of IF (%) Sum of IFs of hostspecific species (%) Sum of IFs of broadhost-range species (%) Number of species 102.2a 21.5b 37.8ab 85.2a 51.1ab 33.3b 58.9 69.3 88.1 50.7a 33.0b 55.2a 78.1 76.3 95.9 17 27 24 24 31 23 31 27 30 Chao2 Jaccard2 Completedness in % 24 25 17 28 50 29 32 18 35 52 59e71 56e72 89e96 60e75 52e55 22 68 26 26 69 45 54 69 27 54 31 29 64 42 56 64 67e83 43e54 77e91 83e94 45e49 51e55 56e58 45e49 38 35 44 41 61e71 73e85 Diversity indices (H0 ) Evenness (E5) 1.78 0.65 2.53 0.77 2.16 0.48 18 1.38 0.36 104 57.0 Chamaecyparis obtusa 16 1.76 0.43 21 2.19 0.60 18 2.75 0.70 29 2.63 0.73 2.46 0.68 2.33 0.64 2.38 0.61 2.00 0.57 2.62 0.63 31 2.15 0.46 2.54 0.56 Letters indicate significant differences among sites (SteeleDwass test, a ¼ 0.05). calculated the similarity of fungal composition among the different host species at each site. The MorisitaeHorn similarity index (CMH) was used to assess similarity in ESTIMATES. X ðani bni Þ=ðda þ dbÞaN bN; CMH ¼ 2 where aN is the total number of isolates in host species A, ani is the number of isolates of the ith species in host species A, bni is the number of isolates of the ith species in host species B, P P da ¼ an2i =aN2 and da ¼ bn2i =aN2 . A Bonferroni correction was applied to the paired nonmetric variance (Wilcoxon signed-rank test) for multiple comparisons of CMH between sites. Nonmetric comparisons by the Table 4 e Dominant fungal endophytes in this study and previous studies (for host code, see Table 3). Present study Previous studies Host species Dominant species Order Host species Qs* Discula sp. Phomopsis spp. Diaporthales Diaporthales Qs Discula sp. Phomopsis sp. Diaporthales Diaporthales Matsuda et al. 2010 Qm*, Qg Tubakia sp. 1 Tubakia sp. 2 (QA-b) Tubakia sp. 3 Diaporthales Diaporthales Diaporthales Quercus acuta QA-b (Tubakia sp.) Discula sp. Phomopsis sp. Diaporthales Diaporthales Diaporthales Hashizume et al. 2008 Ej* C. gloeosporioides Phy. capitalensis R. cerophilum Glomerellales Botryosphaeriales Capnodiales Camellia japonica C. gloeosporioides Geniculosporium sp. 1 C. acutatum Glomerellales Xylariales Glomerellales Osono 2008 Co* Xylaria sp. Xylariales Chamaecyparis lawsoniana Scolecosporiella sp. Pleosporales Petrini & Carroll 1981 Pezicula sp. Helotiales Nodulisporium sp. Geniculosporium sp. Chloroscypha alutipes Pezicula sp. Xylariales Xylariales Helotiales Helotiales Cj* Phy. cryptomeriae Xylaria sp. Botryosphaeriales Xylariales Fj Glomerella sp. Phomopsis spp. C. gloeosporioides Glomerellales Diaporthales Glomerellales Discula sp. Ascochyta fagi Diaporthales Pleosporales Cc Phy. capitalensis Phomopsis spp. C. gloeosporioides Botryosphaeriales Diaporthales Glomerellales Ct Gnomonopsis sp. Phomopsis spp. Diaporthales Diaporthales Fagus crenata Order References Sahashi et al. 2000 Hashizume et al. 2010 Alnus rubra Betula pubescens, B. pendula *Host common to all sites. Dominant species Gnomonia setacea Gnomoniella tubiformis Fusicladium betulae Gnomonia setacea Melanconium betulinum Diaporthales Diaporthales Pleosporales Diaporthales Diaporthales Sieber et al. 1991 Helander et al. 2007 Fungal endophytic diversity in urban forest 197 Table 5 e BLAST search results for major endophytic fungi. Fungal species Phyllosticta cryptomeriae White sterile Cs2 Gonomonopsis sp. White sterile PH1 Tubakia sp. 2 Pezicula sp. Pseudocercospora sp. Tubakia sp. 1 Ascochyta fagi Ramichloridium cerophilum Yellow sterile GL2 Discula sp. 1 Yellow sterile GL1 Tubakia sp. 3 Discula sp. 2 Rosellinia sp. 2 White sterile DT1 Glomerella sp. (Colletotrichum sp. 3) White sterile WM1 Phomopsis sp. 2 Xylaria sp. Botryosphaeria dothidea WHS Phyllosticta capitalensis Colletotrichum acutatum Alternaria sp. Nigrospora sp. 2 Astrocystis sp. Phomopsis spp. White sterile WH7 Aureobasidium sp. Colletotrichum gloeosporioides Muscodor fengyangensis Pestalotiopsis sp. Accession number BLAST search result Accession number Score (%) AB731136 Phyllosticta cryptomeriae AB454271 629/632 (99 %) AB731135 Gnomonopsis sp. JN793536 539/564 (96 %) AB731134 AB731133 Dicarpella dryina Pezicula sp. JF502454 AF141173 531/591 (90 %) 517/531 (97 %) AB731132 QA-b AB365875 522/600 (87 %) AB731131 Ramichloridium cerophilum HQ608156 473/480 (99 %) AB731130 Rosellinia sp. HQ907947 513/519 (99 %) AB731129 AB731128 Glomerella lindemuthiana Fungal endophyte sp. FN566869 AB255246 571/573 (99 %) 510/512 (99 %) AB731127 AB731126 Xylaria sp. Botryosphaeria dothidea HM595549 AB645751 555/555 (100 %) 557/558 (99 %) AB731124 AB731123 Guignardia mangiferae Colletotrichum acutatum AB454332 AJ301905 628/628 (100 %) 542/550 (99 %) AB731121 Astrocystis cocoes AY862571 457/478 (96 %) AB731122 Muscodor fengyangensis HM034852 551/564 (98 %) SteeleDwass test were used to detect differences in IF and the number of isolates of fungi with a broad host range. Relationships between host-specific groups, broad-hostrange groups, and whole fungi were evaluated using the nonparametric Spearman’s correlation test. JMP ver. 9. 03 was used for these statistical analyses. Multiple classification analysis and nonmetric multidimensional scaling (NMS) were performed for 57 fungal communities (units) in the 57 trees using the Sørensen index and 50 randomized runs. Fungal taxa that had a broad host range were grouped according to their host preference through two-way cluster analysis, with their relative dominance based on the IF of each host species in each site. PC-ORD ver. 5 was used for the community analysis. Results 3 % in each sample unit (i.e., host tree species in each site). The host-specificity index (Si) for the 34 frequent morphotypes ranged from 0.43 to 1.00. It consisted of a mixture of four normal distributions. Thus, a value of 0.72, which divided the second and third distributions, was used as the border between host-specific species and species with a broad host range. Overall, seven species in 11 genera with Si values higher than 0.72 were defined as host-specific fungi (Table 2). All tree species had at least one host-specific endophyte species, which were isolated from the specific host species at all sites. Host-specific species were Phyllosticta cryptomeriae and Rosellinia sp. 2 in Cj (for host code, see Table 2), Pezicula sp. and White DT1 in Co, Ramichloridium cerophilum and Yellow GL1e2 in Ej, PH1 in Qg, Tubakia sp. 3 (‘QA-b’ in Hashizume et al. 2008) in Qm and Qg, Tubakia sp. 1e2 in Qm, Discula sp. 1 in Qs and Fj, Discula sp. 2 in Cc, Ascochyta fagi in Fj, and Gnomonopsis sp., Cs2, and Pseudocercospora sp. in Ct. In Fagaceae, six hostspecific species belonged to Diaporthales. Frequency of fungal species and their host specificity IF, fungal richness, and diversity In total, 6071 isolates were detected from 4320 leaf segments taken from the three sites. The isolates were classified into 128 morphotypes. Thirty-four morphotypes (fungal species; Table 5) were distinguished as major taxa, and the other 94 morphotypes were defined as rare fungi, whose IF was under The sum of the IFs for all fungi in the hosts Co, Qm, and Qs was significantly lower in the suburban forest than in the two rural forests. The other two host species exhibited the same trend, although the difference was not significant (Table 3). The sum of the IFs of host-specific species in Ej, Qm, and Qs was significantly 198 E. Matsumura, K. Fukuda lower in the suburban forest than in the rural forests, and the other two host species also exhibited the same trend (Table 3). Moreover, the sum of the IFs of species with a broad host range in Cj, Co, and Qm was also significantly higher in the rural WG site and was lowest in the suburban forest. In Cj and Co, the sum of the IFs of species with a broad host range exhibited strong positive correlations with the sum of the IFs of all fungal species (Cj, 0.72, Co, 0.78; Spearman’s correlation coefficient, P < 0.0001). On the other hand, in Co, Ej, Qm, and Qs, the sum of the IFs of species with a broad host range exhibited negative correlations with the sum of the IFs of hostspecific species (Co, 0.45, Ej, 0.63, Qm, 0.45, Qs, 0.62; P < 0.02). Although the number of fungal species and fungal diversity (H0 ) did not differ significantly among sites for each host species, species accumulation (rarefaction) curves indicated that fungal richness and the number of isolations were lowest in the suburban forest (Fig 5). variation, and the fungal communities were separated according to host species and site (Fig 2; Stress ¼ 14.1, P < 0.02). Fungal communities on the same host displayed less variation; however, fungal communities on different hosts at the same site were sometimes less varied than those on allopatric conspecific hosts (e.g., Qm and Ej at KS and WG; Cj and Co at WG). Fungal communities were more strongly influenced by sympatry on some hosts or in some sites. To assess the similarity of fungal communities among tree species within a site, we calculated the volume of a polyhedron formed by five points consisting of five tree species common to all three sites. The volumes were 0.58 at WG, 0.70 at TK, and 0.36 at KS (Fig 2), which suggested that the fungal communities among tree species in the suburban forest were most similar. This result was confirmed by the CMH among sites, which was significantly higher for KS than for TK (paired nonparametric Wilcoxon signed-rank test, a < 0.05, Bonferroni correction). Fungal communities Fungi with a broad host range To summarize and visualize the compositional differences in fungal communities among host species and sites, we used NMS. Three axes accounted for a substantial 78.5 % of the Chamaecyparis obtuse A1 C C. acutatum C. gloeosporioides Quercus myrsinaefolia 3 C. sp.3 Eurya japonica 4 Phy. capitalensis Quercus serrata 5 Phomopsis spp. 6 Phomopsis sp.2 7 WM1 8 Pestalotiopsis sp. 9 Nigrospora sp.2 Quercus glauca Carpinus tschonoskii 0 1 2 Fagus japonica Axis 3 (42.5%) 1.5 Cryptomeria japonica Castanea crenata Mt. Takao Mt. Wagakuni Kashiwa Qm Qm Axis 3 (42.5%) 1.5 Fungal taxa with a Si lower than 0.72 were defined as species with a broad host range. They were not always isolated from Fj Ej Ej Qm 4 1 3 2 Cc 0 12 7 6 5 Qs 10 A. cocoes 8 11 910 13 Ct Ct 11 WH7 Co 12 M. fengyangensis 13 Xylaria sp. Co Cj Cj -1.5 -1.5 0 -1.5 Axis 2 (24.1%) 1.5 1.5 Axis 2 (24.1%) 1.5 B1 0 B2 Axis 3 (42.5%) A2 Axis 3 (42.5%) 0 -1.5 1.5 D Qm Qm Ct Ct Fj 14 Qm 6 5 0 Ej Ej 3 2 Cc 7 Qs 12 11 10 8 9 Co Co 13 Cj Cj -1.5 -1.5 0 Axis 1 (11.8%) 1.5 -1.5 -1.5 0 1.5 Axis 1 (11.8%) Fig 2 e Nonmetric multidimensional scaling (NMS) of fungal species isolated in nine tree species at three sites. Each figure represents a sample unit (A, B) or fungal species (C, D). The letter indicates the specific host species in C, D. The similarity of fungal communities is compared for three sites (A-1, B-1). Fungal endophytic diversity in urban forest 100 B C D Similarity (%) 50 75 100 25 0 80 Phomopsis spp. Phomopsis sp.2 Alternaria sp. WM1 Astrocystis sp. WHS C. gloeosporioides C. acutatum P. capitalensis Glomerella sp. Xylaria sp. M. fengyangensis Pestalotiopsis sp. Nigrospora sp.2 WH7 B. dothidea Aureobasidium sp.2 Number of species A 199 60 40 WG 20 TK KS 0 Fig 3 e Isolation trend for broad-host-range fungi by cluster analysis. 0 200 400 600 800 1000 Number of isolates Isolation frequency (%) Fig 5 e Rarefaction curves of the endophytic fungi in three sites for five common host species. WG: Mt. Wagakuni; TK: Mt. Takao; KS: Kashiwa. 150 a a 100 b b 50 a a b b b C D A b b b 0 A B Deciduous B C D A Evergreen B C D Conifer Mt. Wagakuni 150 a 100 b 50 a b b c b c 0 A B C D Deciduous A B C D A Evergreen B C all sites. The dendrogram resulting from the two-way cluster analysis (Fig 3) at 30 % similarity produced four main clusters, which were characterized by the following fungal groups: cluster A, Phomopsis spp., frequently isolated from deciduous broadleaves; cluster B, Colletotrichum spp. from evergreen broadleaves; cluster C, Xylariaceae; and cluster D, Pestalotiopsis sp., from conifers in WG and KS. These four groups of fungi with a broad host range are shown in Fig 4, and their distribution varied according to the host-species life forms. This trend was consistent for NMS fungal plots (Fig 2C and D). We compared the number of isolates in the four clusters among sites. At WG, where conifers and evergreens were sympatric, cluster C fungi were more frequently found in evergreen hosts. At TK, which was dominated by evergreen trees, fungi belonging to cluster B showed a higher frequency in deciduous hosts than at other sites. At KS, a mixed evergreen and deciduous forest, cluster A in evergreen and cluster B in deciduous hosts were more frequent than in other sites. Cluster D in deciduous hosts was more frequent than cluster C at KS. D Discussion Conifer Mt. Takao Host specificity 150 100 a a a 50 b b c c b D A c ab ab a B D 0 A B C D Deciduous A B C Evergreen C Conifer Kashiwa Fig 4 e Boxewhisker plots comparing ranked medians of IF of fungal groups with a broad host range in nine tree species at three sites. Cluster A, grey; cluster B, open; cluster C, dark grey; cluster D, slashes, see Fig 3. Different letters indicate significant differences among the four fungal groups (SteeleDwass test, a [ 0.05). To identify differences in fungal endophyte communities between suburban and natural forests, we investigated the fungal diversity of suburban and rural forest sites in Japan and considered the endophytic communities in conspecific tree leaves. All host species studied had one to three hostspecific species, which often dominated in their host. When compared to previous studies of the same or closely-related hosts worldwide, many common or similar fungal taxa were shown to be dominant regardless of geographical differences (Table 4). Our study is the first detailed report of endophytic fungal communities and their host tree species, with the exception of Quercus serrata. For Q. serrata, the dominant fungi (Discula sp. and Phomopsis sp.) were the same as those identified by Matsuda et al. (2010) in western Japan. Endophytic communities in Fagaceous and Betulaceous trees were dominated by Diaporthales. Quercus spp. in particular exhibited 200 a high affinity with Diaporthales. On the other hand, communities in Fagaceae, except for the Quercus genus, were frequently dominated by Colletotrichum spp. and Phyllosticta capitalensis, which are recognized as common fungi of evergreen hosts. This may suggest that forest type also affects endophytic dominance. In tropical forests, Suryanarayanan (2011) showed that the infection frequency of Phomopsis spp. gradually decreased when forests became wetter and the infection frequency of Colletotrichum spp. increased with increasing annual rainfall before reaching its maximum in an evergreen forest that received the greatest rainfall of the forests studied. In our study, Colletotrichum spp. and Phy. capitalensis were more frequently isolated from evergreen trees, and Phomopsis spp. were more frequently isolated from broadleaved trees (Fig 4). Our results are consistent with Suryanarayanan (2011) in terms of the combinations of host life forms and fungal genera. This might suggest that their abundance could be influenced by the life form of the dominant hosts (deciduous or evergreen) rather than rainfall or humidity. Differences in rainfall and humidity among our study sites were almost negligible compared to Suryanarayanan (2011). Additionally, Xylariales were frequently isolated from two conifer species (Fig 4), which supports the findings of a previous study of Cupressaceae € ller 1979; Petrini & Carroll 1981; Bills & (Petrini & Mu Polishook 1992; Hoffman & Arnold 2008). Therefore, we conclude that these fungi with a broad host range also have some host preferences. Decrease in endophyte diversity in suburban forest (KS) In the suburban forest, the IF values for endophytes as a whole and for host-specific fungi were both lower than in rural forests. The decrease in host-specific fungi and the increase in fungi with a broad host range resulted in the highest CMH being observed between tree species in the suburban forest, namely, a low b diversity of endophytic communities in suburban forest. In the same Kanto area, Ochimaru & Fukuda (2007) compared a mushroom community across a gradient from urban to rural areas and demonstrated that species richness and the frequency of ECM fungi were negatively correlated with urbanization. Urbanization can cause not only microclimate changes, including heat-island effects, and chemical changes because of pollution, but also forest fragmentation and isolation. However, previous reports have not referred to forest isolation. Helander et al. (2007) studied leaf endophytes in Betula spp. in an archipelago in Finland and showed that total isolation frequencies of endophytes decreased with increasing distance from the mainland and decreasing size of the island. Our study was consistent with this result if we regard the fragmented forests in a suburban area as islands in a city. Furthermore, Drinnan (2005) clarified fragmentation thresholds by urbanization for multibiota with different trophic levels. The study indicated a potential threshold in a reserve of approximately 2 ha in size, below which plant and fungal species richness decreased rapidly. The suburban forest in our study was approximately 1.5 ha in size and was also isolated from other forests (Fig 1). Although fungal species richness and the diversity index per single host species (a diversity) among the three sites did not E. Matsumura, K. Fukuda differ, the lower total IF and lower b diversity in KS could be attributed to limited spore dispersal in fragmented forests. Positive relationships between host density and the frequency of dominant endophytic fungi, especially in host€ ller & Hallaksela specific species, have been reported (Mu 1998; Helander et al. 2006). In our study, Quercus serrata and Quercus myrsinaefolia were dominant in the suburban forest; however, the frequency of their dominant endophytes, Discula sp. and Tubakia spp., was lower than in the rural forests. This may be because the number of tree species belonging to the genus Quercus was lowest in the suburban forest (one evergreen species and two deciduous species at KS; two and two at WG; seven and three at TK), and the total dominance of these oak species (i.e., an infection source) in the suburban forest was the lowest of the three sites. This trend was also observed in other host species. There are many possible explanations for the difference in endophytic communities between suburban and rural forests. Both urbanization and forest fragmentation can lead to decreases in the diversity of endophytic communities. The experimental design used in this study does not allow us to distinguish the effects of fragmentation from other factors, such as microclimatic changes or pollution; however, these various influences of urbanization on host-specific fungi and fungi with a broad host range were observed not only in this study but also in other urban forests (E.M. & K.F., unpubl. data). These declines in host-specific fungi in urban forests support our hypothesis that forest fragmentation and isolation in urban areas affect the endophytic community. In conclusion, the endophytic community in temperate forests is characterized by high host specificity, namely high b diversity among host species. The infection rate of fungi with a broad host range also exhibits some host preference. However, in isolated suburban forests, the number of hostspecific endophytes decreases, and the frequency of fungi with a broad host range increases. Endophytic communities in suburban forest have been shown to be depauperate, with low b diversity. This may be a result of the decreased infection source of host-specific fungi in isolated urban forests. Acknowledgement The authors thank Dr Y. Hashizume for help with the sampling design and sampling. We also thank Dr Y. Yaguchi for fungal identification and Dr N. Sahashi, Dr T. Umebayashi and Dr A. Oda-Tanaka for their advice and support. 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