A comparison of the community diversity of foliar fungal endophytes between seedling and adult loblolly pines (Pinus taeda)

Accepted Manuscript A comparison of the community diversity of foliar fungal endophytes between seedling and adult loblolly pines (Pinus taeda) Ryoko Oono, Emilie Lefèvre, Anita Simha, François Lutzoni PII:

S1878-6146(15)00120-8

DOI:

10.1016/j.funbio.2015.07.003

Reference:

FUNBIO 602

To appear in:

Fungal Biology

Received Date: 17 April 2015 Revised Date:

14 June 2015

Accepted Date: 3 July 2015

Please cite this article as: Oono, R., Lefèvre, E., Simha, A., Lutzoni, F., A comparison of the community diversity of foliar fungal endophytes between seedling and adult loblolly pines (Pinus taeda), Fungal Biology (2015), doi: 10.1016/j.funbio.2015.07.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A comparison of the community diversity of foliar fungal endophytes between seedling and adult loblolly pines (Pinus taeda). Ryoko Oono1,2

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Emilie Lefèvre1 Anita Simha1 François Lutzoni1

Department of Biology, Duke University, Durham, NC, USA; 2Current Address: Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, CA, USA

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_____________________________________ Corresponding author: Ryoko Oono, PhD Ecology, Evolution, and Marine Biology University of California – Santa Barbara Santa Barbara, CA 93106 Phone: 805-893-5064 Fax: 805-893-2266 [email protected]

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3/20/15 12:32 PM ABSTRACT Fungal endophytes represent one of the most ubiquitous plant symbionts on Earth and are

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phylogenetically diverse. The structure and diversity of endophyte communities have been shown to depend on host taxa and climate, but there have been relatively few studies exploring endophyte communities throughout host maturity. We compared foliar fungal endophyte

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communities between seedlings and adult trees of loblolly pines (Pinus taeda) at the same

seasons and locations by culturing and culture-independent methods. We sequenced the internal

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transcribed spacer region and adjacent partial large subunit nuclear ribosomal RNA gene (ITSLSU amplicon) to delimit operational taxonomic units and phylogenetically characterize the communities. Despite the lower infection frequency in seedlings compared to adult trees, seedling needles were receptive to a more diverse community of fungal endophytes. Culture-free method confirmed the presence of commonly cultured OTUs from adult needles but revealed

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several new OTUs from seedling needles that were not found with culturing methods. The two most commonly cultured OTUs in adults were rarely cultured from seedlings, suggesting that

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host age is correlated with a selective enrichment for specific endophytes. This shift in endophyte species dominance may be indicative of a functional change between these fungi and

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their loblolly pine hosts.

Key words: Class 3 endophytes, fungal communities, phylogeny, Pinus taeda, plant-fungal symbiosis

INTRODUCTION Plants harbor numerous and phylogenetically diverse species of endophytic fungi without symptoms of disease. Those that are horizontally transmitted among hosts with localized

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infections in above ground tissues (Class 3 endophytes, sensu Rodriguez et al. [2009]) are especially diverse and their ecological roles remain largely unknown. A number of studies have suggested that some endophytic species have beneficial effects on their hosts, including pathogen

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defense (Minter 1981; Arnold et al. 2003), herbivore resistance (Diamandis 1981; Carroll 1988b; Miller et al. 2008), as well as heat and drought tolerance (Bae et al. 2009). In return, it is

assumed that endophytic fungi benefit from the interaction by acquiring protection and nutrition

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from their hosts and, in many cases, reproducing sexually on dead tissues of their host plant (Carroll & Carroll 1978; Saikkonen et al. 1998). However, the nature of the plant-endophyte

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symbiosis likely falls along a mutualism-parasitism continuum (Saikkonen et al. 1998; Sieber 2007) depending on the host-endophyte genotype-genotype interaction, environmental context, and the state of the host health (Carroll 1988a; Redman et al. 2001), for example. Exploring endophyte species diversity and composition across environmental gradients and host contexts

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will help identify endophytic species with ecologically distinct roles as well as present a useful means to understand the environmental variables responsible for structuring fungal diversity. Comparisons of endophyte communities across host age, i.e., seedling to adult stage,

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have been rarely conducted (although see Ferreira Rodrigues 1994), whereas comparisons over leaf age (young to old leaves) are more commonly explored (Espinosa-Garcia & Langenheim

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1990; Ferreira Rodrigues 1994; Frohlich et al. 2000; Arnold & Herre 2003;). Comparison of species abundance and richness across host tissue types is also limited in studies of foliar fungal endophytes with proper molecular data (although see Sandberg et al. 2014). Aspects of fungal endophyte communities that are unique to different life stages of a host are likely to give clues about host specificity, coevolution, host mechanisms for recruiting horizontally transmitted 2

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endophytic fungi as well as, potentially, any beneficial effects on host fitness, which are challenging to assess experimentally for endophytic fungi (Sieber 2007). The goal of this study was to compare communities of foliar fungal endophytes in adult

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trees vs. seedlings of loblolly pines (Pinus taeda) in North Carolina’s Duke Forest and to identify the most common and recurring endophyte species at these two different stages of host

development. Foliar fungal communities were sampled during two seasons (summer and winter)

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for both adult trees and seedlings with culturing methods. Sampling was supplemented with culture-independent cloning to identify unculturable or slow-growing endophytic fungi. We used

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both taxonomy-dependent and operational taxonomic unit (OTU)-based approaches to characterize the fungal endophyte communities using the nuclear ribosomal internal transcribed spacer (ITS) and adjacent partial large subunit (nrLSU) RNA coding region. Exploring fungal endophytes in P. taeda of the Duke Forest using these molecular markers also allowed us to

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compare our results with a past endophyte diversity study of P. taeda by Arnold et al. (2007).

MATERIALS and METHODS

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Sampling pine needles and tissue processing

In 2010, the Free-Air CO2 Enrichment (FACE) project in the Blackwood Division of

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Duke Forest (Orange County, NC) was completed (Hendrey et al. 1999) and half of each experimental plot was deforested. This led to the natural regeneration of pine seedlings in the newly opened half of each plot adjacent to adult P. taeda trees that were twenty to thirty years old. Three of the plots that had previously received ambient CO2 treatments (1, 5, and 6) were targeted for sampling. 3

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Field sampling was conducted in summer (June-August 2012) and winter (December 2012 – January 2013), representing four sampling treatments; adult-summer, adult-winter, seeding-summer, seedling-winter, respectively. At each of the three plots, 11 to 12 of the oldest

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second-year needles from three adult trees (total of 9 trees), were sampled using tree pruners or by climbing observation towers from multiple branches at various compass directions between 6 and 8 meters above the ground. Second-year needles are distinguished from first-year needles by

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the color of their fascicle sheaths. For seedlings, we increased the sampling to six individuals per plot (total of 18 seedlings) due to lower isolation frequency compared to adult needles and

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sampled 11 to 20 needles per seedling. The seedling needles all consisted of the oldest primary juvenile needles growing near the lower part of the seedling, which are not fascicled and which are between three and four centimeters long (Bormann 1956). By sampling the oldest needle tissues from both adult trees and seedlings, we were able to compare the most established fungal

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endophyte communities from the respective age classes.

Needles were surface-sterilized by briefly immersing them in 95% ethanol, followed by 2 min in 0.05% hypochlorite and 2 min in 70% ethanol. Two random 2 mm sections distributed

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across the needle length were cut and placed on 2% malt extract agar (MEA) slants in 1.5 ml centrifuge tubes under sterile conditions. A total of 1,284 and 408 needle segments from

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seedlings and adult trees (Table 1), respectively, were allowed to incubate on MEA, a growth media amenable to a broad group of fungal species (Arnold et al. 2007), for at least two months before cultures were sampled for genotyping. The culturing study was complemented with culture-free sampling where pine needles from adult trees and seedlings were collected in June of 2013, surface-sterilized, cut into sections in the same manner as for cultured samples and then 4

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bulked by host age. The culture-free method revealed whether the culture-dependent approach missed fungal species that are abundant in the communities of the two age classes.

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Genotyping fungal endophytes

For cultured fungal endophytes, at least fifty randomly selected isolates from each

sampling treatment were initially processed for genotyping. Small pieces of mycelium (ca. 1 mm

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x 1 mm) were placed in 0.6 ml tubes with 50 µL of glass beads (400 µm VWR silica beads) and 50 µl of Tris-EDTA buffer. DNA was extracted by vortexing the tubes at maximum speed for

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two minutes. Products were diluted with 200 µL of TE buffer for direct use in PCR. We amplified the nuclear ribosomal internal transgenic spacers (ITS1, 5.8S nrRNA, ITS2), and partial 28S large subunit (nrLSU) RNA regions using the primers ITS1F (Gardes & Bruns 1993) and LR3 (Vilgalys & Hester 1990) following PCR protocol outlined in Arnold et al. (2007) with

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an initial denaturation step of 95°C for 4 min, followed by 35 amplification cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 90 s, and a final incubation for 10 min at 72°C. PCR products were cleaned with ExoSAP-IT (Affymetrix) and

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sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) followed by analysis with an Applied Biosystems 3730xl DNA Analyzer. The ITS1 or ITS2

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region is typically used for environmental DNA barcoding (Schoch et al. 2012; Bazzicalupo et al. 2013) and diversity estimation (Nilsson et al. 2008; U'Ren et al. 2009) for fungi, while the nrLSU region has been one of the most commonly used markers for phylogenetic inferences in Fungi (Moncalvo et al. 2000; Arnold et al. 2007). For adult samples, due to low OTU diversity that was detected in a preliminary survey, the number of genotyped cultures was increased by 5

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matching restriction fragment length polymorphism (RFLP) patterns of fifty extra random isolates from each season with those previously sequenced. The ITS-LSU region was amplified and subjected to restriction enzyme digestion by AluI and MspI. All ITS-LSU amplicons with

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unique RFLP bands were sequenced.

For cloning, samples from different plots were bulked by host age and ground under liquid nitrogen with mortar and pestle. DNA was extracted using Qiagen DNeasy kit (Qiagen)

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and the ITS-LSU region was amplified as previously described, but at two annealing

temperatures of 50°C and 52°C, because different annealing temperatures are known to reveal

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different microbial community compositions (Schmidt et al. 2013). PCR products were ligated to a cloning vector with the TOPO PCR cloning kit (Life Technologies). Forty-eight fungal clones from both host ages at each annealing temperature were genotyped using RFLP markers. Clones with unique RFLP bands were sequenced. Three to seven clones or isolates of each RFLP group

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were sequenced and found to be >99% identical within a group, validating the reliability of RFLP markers.

Sequencing for both cultured isolates and clones used a two-step process. Samples were

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first unidirectionally sequenced from the 5’ direction to cover the most variable ITS region using ITS1F. Unique isolates (<99.6% similarity) based on the ITS region were resequenced from the

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3’ direction with LR3. Some sequences were removed due to poor quality. Sequencher 5.0 was used to call bases and assemble reads into consensus sequences and determine sequences with unique ITS regions.

Clustering and phylogenetic analyses 6

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The ITS2 sequences were used to delimit operational taxonomic units (OTUs) using 90%, 95%, 97%, and 99% sequence similarity (Suppl. Table 1). A 95% grouping criterion is commonly used to approximate fungal species boundaries (Arnold & Lutzoni 2007) but ITS

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variation is often clade-dependent (Nilsson et al. 2008). Hence, the conservative 99% grouping was used in all following phylogenetic and multivariate community analyses (Pitkaranta et al. 2008). Results are also reported with and without singletons. Sequence clustering was performed

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with UCLUST, which is a de novo clustering approach without a reference database (Edgar 2010) in QIIME (Caporaso et al. 2010).

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We chose one representative sequence from each ITS2 99% OTU cluster and used its linked partial LSU sequence to build our phylogenetic tree. The LSU sequences were aligned with reference sequences collected from GenBank, which included 149 representatives from all major classes within the Ascomycota and Basidiomycota (Suppl. Table 2). References were

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chosen to help identify OTUs to the class level with high bootstrap support. We also included LSU sequences of 29 endophytic isolates and 17 endophytic environmental clones from Arnold et al. (2007)’s study on adult loblolly pine needles sampled in 2004 (Suppl. Table 3). LSU

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sequences were manually aligned in MacClade 4.08a (Maddison & Maddison 2005) and ambiguous regions were identified using the LSU secondary structure model for Saccharomyces

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cerevisiae (Cannone et al. 2012).

We constructed LSU phylogenies using RAxML (Stamatakis 2006; Stamatakis et al.

2008) implemented within SNAP Mobyle workbench (Price & Carbone 2005) with three sequence data sets: i) unambiguously aligned sites only, ii) unambiguously aligned sites with five ambiguously aligned regions recoded with PICS-Ord (Luecking et al. 2011), and iii) 7

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unambiguously aligned sites and ambiguously aligned regions recoded with PICS-Ord with a conservative backbone constraint tree, which consisted of 38 reference taxa based on several published multigene phylogenies (Spatafora et al. 2006; Wang et al. 2006; Schoch et al. 2009;

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Prieto et al. 2013; Machouart et al. 2014; Suppl. Table 2 and Suppl. Fig. 1). Alignments with and without delimited ambiguous regions are deposited in TreeBASE (accession XXX). The results of the nonparameteric bootstrap analyses were based on 1000 pseudoreplicates.

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We report the potential identities of some endophyte OTUs based on high

max identity

95%,

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similarities of ITS2 or ITS1 to known species using BLAST (criteria: query cover

95%, published in peer-reviewed journals; Suppl. Table 4). However,

inferring species identities with BLAST has many limitations and should only be

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community.

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considered as a temporary reference to understand the species diversity of a

Multivariate community analysis

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We compared the composition of fungal OTUs between needles from adult trees and

seedlings by performing hierarchical clustering analyses in UniFrac (Lozupone et al. 2006) with the ML tree built using ambiguous regions coded by PICS-Ord and the conservative backbone constraint tree. We performed each analysis with or without singletons as well as weighted or unweighted with abundance data, assigning more weight on abundant or rare lineages, 8

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respectively. The communities were analyzed in groups of equal numbers of individuals (3) per age category, which correspond to whole plots for adult samples and half of each plot for seedling samples. We assessed the robustness of the clusters at the smallest sample size within a

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group using jackknife analysis with 1000 permutations in the UniFrac framework. UniFrac

distances between pairs of groups were also used as input for principal coordinates analysis to illustrate the variation among the fungal endophytic communities in different host ages and

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seasons.

Rarefaction curves, extrapolations, and bootstrap estimates of total OTU richness were

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inferred between host age communities identified by culturing using EstimateS v9.1.0 (Colwell 2013). Rarefaction curves were extrapolated to 2x the number of genotyped isolates, beyond which the variance increases greatly. Bootstrap estimates were inferred with 100 randomizations of sample order. Rarefaction curves using 95% and 90% ITS2 similarity criteria are reported in

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the supplemental files (Suppl. Fig. 2). Alpha diversity was calculated in the R package vegan with a parametric estimator, Fisher’s alpha, and a non-parametric Simpson’s diversity index. Diversity indices were calculated for the entire community for each age as well as per group of

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three individuals within each age category. Community samples were also randomized and subsampled without replacement to yield 1000 random partitions of 48 sequences each. The

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distributions of diversity indices from the random partitions were compared between adult and seedling culture samples with t-tests as well as against the observed values from respective cloning samples with one-sample t-tests. We excluded Fisher’s alpha values over 1000, characteristic of small sample sizes with high species richness, from statistical analyses. The Wilcoxon sign-rank test was used to test if certain fungal endophyte OTUs were more common 9

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in one host age than the other by comparing the isolation frequencies of the most commonly isolated OTUs from seedling and adult individuals from the same plots.

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RESULTS

Pinus taeda seedlings yielded endophytic fungal growth from 180 out of 1,284 needle segments (14.0%), whereas leaves from adult trees yielded growth from 313 out of 408 needle

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segments (76.7%). The isolation frequencies differed significantly between seedlings and adult trees (t = 9.23, df = 6.68, p < 0.01). Random fungal isolates were genotyped from seedlings (57

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winter and 50 summer; 107 total) and adult trees (95 winter and 97 summer; 192 total; Table 1). We sequenced 96 fungal clones from both adult trees and seedlings to identify any endophytic OTUs that may not be detected by our culturing method. A total of 491 isolates and clones were genotyped (Table 1) of which 182 were sequenced bidirectionally with ITS1F and LR3 primers

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(GenBank accession KM519195-KM519376; Suppl. Table 4 & 5). RFLP matched 170 isolates and clones as one of eleven OTUs (Table 1, Suppl. Figure 3, Suppl. Table 4).

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Clustering and phylogenetic analysis

Based on 99% ITS2 similarity, 299 cultured isolates clustered to 90 OTUs. Communities

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from seedlings and adult trees shared 16 OTUs (Fig. 1). Seedlings had 51 OTUs not found in adults whereas adults had 23 OTUs not found in seedlings. In both seedlings and adults, the majority of non-overlapping OTUs were singletons, 82.4% (42/51) and 73.9% (17/23), respectively. Since the OTU communities between seasons were not structurally significant for

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either seedlings or adult trees (Fig. 2, Suppl. Figure 4a, c), they were combined and analyzed together hereafter. Based on 99% ITS2 similarity, 192 clones clustered to 39 OTUs. Only one OTU was

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shared between seedlings and adult trees (Fig. 1). About half of non-overlapping OTUs were singletons for the cloned communities, 51.5% (17/33) for seedlings and 40.0% (2/5) for adults. Both culturing and cloning detected far more singletons in the seedlings than in adults

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(Fig. 1). In total, culturing and cloning methods identified 118 ITS2 OTUs, of which 17

overlapped between seedling and adult endophyte communities, which correspond to 18.1%

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(17/94) and 41.5% (17/41) OTUs from the respective communities. Cloning revealed 28 (71.8% of total cloned OTUs) more OTUs that were not found by culturing, whereas culturing revealed 79 (87.8% of total cultured OTUs) more OTUs that were not found by cloning. Out of 118 ITS2 OTUs, 83 (70.3%) had high ITS similarity to known species, 27 (22.9%)

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had high similarity to unnamed sequences, and 8 (6.8%) had no high similarity in GenBank (Suppl. Table 4). The final dataset for the phylogenetic analysis consisted of 454 base pairs of

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the LSU from 149 reference sequences (Suppl. Table 2), 46 sequences from Arnold et al. (2007; Suppl. Table 3), and 117 OTUs from this study (Suppl. Table 4). Two OTUs (e50ss002

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and e50ss004) that grouped in different OTUs based on ITS2 clustering had identical LSU sequences and were merged, thereby condensing the original 118 OTUs to 117. Out of 454 LSU nucleotides, 292 were unambiguously aligned and five ambiguous regions were identified (positions 24-39, 63-92, 133-157, 341-411, 431-450), resulting in 11

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162 ambiguous sites being coded by PICS-Ord. Major classes of Ascomycota and Basidiomycota were resolved as monophyletic and typically with high bootstrap support

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( 70%), except the Dothideomycetes (Fig. 3). Although the Dothideomycetes was not resolved as monophyletic (here forming a polytomy with Arthoniomycetes), all of our

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OTUs within this class grouped with known Dothideomycetes species with high support.

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The OTUs detected by our study were distributed across four classes within the Basidiomycota and six classes within the Ascomycota (Fig. 3). One OTU (e50ss030) could not be placed within a known fungal class, but a follow-up was not pursued since the OTU was a singleton identified by cloning. The most commonly cultured OTU from adult trees, sampled

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during the summer and winter (caw010; 38.1% and 23.2%, respectively), was resolved within the Atractiellomycetes (Basidiomycota) and had no high similarity to BLAST searches. The second most commonly cultured OTU from adult trees, for both summer and winter (cas039;

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35.1% and 17.9%, respectively), had 99% ITS2 similarity to the Dothideomycetes species Septorioides pini-thunbergii (Quaedvlieg et al. 2013). In contrast, the most commonly isolated

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OTUs from seedlings were from the Sordariomycetes (e.g., csw046, css003, csw043, csw026; Table 2). However, no single Sordariomycetes OTU was found more than 9.3% of the time at either host age. The Sordariomycetes had the greatest OTU richness from both adult tree and seedling communities, with 53 OTUs overall (44.9%) and 35 of these (35/53; 66.0%) being singletons.

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Cloning identified OTUs in three more classes (Exobasidiomycetes, Tremellomycetes, and Lecanoromycetes) compared to what was found with culturing (Fig. 3, Table 2). More Dothideomycetes were detected by cloning than any other class for both seedling and adult

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fungal communities, although this was mostly due to the overrepresentation by one OTU cas039. For clones obtained from adult trees, three common OTUs (caw049, caw010, and

cas039) made up the majority of the community and only three other OTUs were detected (Fig.

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Multivariate community analyses

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3, Table 2).

Endophyte communities in needles of adult trees were highly similar to each other compared to those of seedling needles. The jackknife analysis supported the separate clustering of fungal communities from adult trees vs. seedlings (>99%), except in the case of one adult

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sample or one seedling sample (Fig. 2), which tended to cluster in the other group depending on whether the analysis weighted or unweighted abundance data, respectively. Results were similar with and without singletons (data not shown).

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Based on 99% ITS2 similarity, OTU diversity was greater for seedlings than for adult trees (67 OTUs/107 isolates vs 37 OTUs/192 isolates; Fisher’s alpha = 76.7 vs. 13.6; Simpson’s

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index = 3.9 vs. 2.4, respectively). Fisher’s alpha was statistically significant between adult and seedling communities per three individuals (t = 3.4, df = 8.5, p < 0.01) but not significant for Simpson’s index (t = 1.9, df = 6.1, p = 0.11). Randomization analyses for partitions of 48 cultured isolates indicated that the richness was significantly different for both Fisher’s alpha (t = 89.5, df = 1021.3, p < 0.001) and Simpson’s index (t = 141.9, df = 1075.9, p < 0.001; Suppl. Fig. 13

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5). OTU diversity was also greater with environmental cloning for seedlings than for adult trees (34 OTUs/96 clones vs. 6 OTUs/96 clones; Fisher’s alpha = 18.8 vs. 1.4; Simpson’s index = 0.9 vs. 0.6, respectively). Randomization analyses of cultured samples indicated that the Simpson’s

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index and Fisher’s alpha values for cloning were both significantly lower than expected from culturing alone (Suppl. Figure 5).

Rarefaction curves for the fungal OTUs detected in adult P. taeda approached a plateau

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but did not for seedlings (Fig. 4). The 95% confidence interval for the endophyte community from adult trees remained lower than the observed richness of endophyte OTUs in the endophyte

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community from seedlings.

To test whether particular OTUs associated more with needles from adult trees or seedlings, we compared the frequency of the three most common OTUs from each host age by the Wilcoxon sign-rank test by pairing adult and seedling communities from the same plots. We

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found that two of the OTUs, caw010 and cas039, were significantly more represented in adult communities than in seedlings (Table 3).

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DISCUSSION

This study compares the fungal endophyte communities between seedlings and adult

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trees of P. taeda using molecular data and identifies fungal OTUs that are common vs. unique to these two age categories to better understand fungal endophyte specialization. We found that the two most commonly isolated endophytic fungal OTUs from adult trees, an unknown species from the class Atractiellomycetes (caw010) found 26.6% of the time and a species tentatively identified as Septorioides pini-thunbergii (cas039) found 30.7% of the time, which were also 14

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found by Arnold et al. (2007), were found rarely in needles of seedlings as cultures (Table 3). This suggested that these species are specialized to adult needles and the canopy environment compared to needles of seedlings and the environment close to the soil. The most abundantly

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found OTUs from seedlings (Table 2) were not isolated significantly more from seedlings than from adult trees (Table 3). Furthermore, most of the endophyte OTUs from seedlings were only found once (Fig. 1). The high OTU richness in the seedlings may be due to seedlings having

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little to no specificity to most fungal endophytic species, thereby becoming vulnerable to a diverse group of fungal species, endophytic, pathogenic, and saprotrophic species alike,

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compared to adult needles. Alternatively, a more exhaustive sampling might reveal that the OTU richness from needles of seedlings and adult trees are not significantly different. However, differences in the relative abundances of certain OTUs are clear between the two host age categories, suggesting that some endophyte OTUs may specialize on adult tissues and have

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distinct ecological roles from other endophytes. Whether this is mainly due to differences in innate susceptibility between adult trees and seedlings or differences in selection by the correlating environmental factors, such as the inoculum community, proximity to the canopy or

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soil, microclimatic factors, or life spans of different leaf types, is still unknown. Isolation frequency for foliar fungal endophytes increases with leaf age or exposure

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duration (e.g., Ferreira Rodrigues 1994; Kumaresan & Suryanarayanan 2002; Arnold & Herrera 2003) whereas an increase with host age has not been previously observed (Espinosa Garcia & Langenheim 1990; Ferreira Rodrigues 1994). In this study, we found that seedlings that were not under a canopy had considerably lower isolation frequency than leaves of adjacently growing adult P. taeda. Since leaf age was not controlled between the seedlings and adult trees, it is 15

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possible that seedling needles were younger than needles collected from adult trees even though we sampled the oldest healthy needles available from seedlings at the time. However, despite the possible younger leaf age of seedlings, we found greater OTU richness as well as alpha diversity

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in the endophyte community from seedlings than from adult needles. Endophyte studies

comparing species richness across leaf ages have mixed findings from no difference (Frohlich et al. 2000), an increase (Kumaresan & Suryanarayanan 2002), to an initial increase and then

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decrease in species richness (Espinosa Garcia & Langenheim 1990) with increasing leaf age. Espinosa Garcia & Langenheim (1990) found that endophyte diversity tended to increase from

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one to three-year-old leaves of coastal redwoods (Sequoia sempervirens) and then decrease in fully mature needles that were eight years or older. They also found that the young tissues of redwood sprouts from the base of trees tended to have higher diversity than the trees, which had a more uneven community with several dominating fungal species. However, Ferreira Rodrigues

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(1994) compared fungal endophyte communities between saplings and mature trees of Amazonian palm (Euterpe oleracea) and found no difference in diversity. It should be noted that these previous studies comparing fungal endophyte communities across leaf or host ages were

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based on morphological data from cultures only and hence, have limited inference in estimating species richness (Arnold et al. 2007). However, Espinosa Garcia & Langenheim (1990)’s study

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finds similar patterns as ours, suggesting that a decrease in species diversity of fungal endophytes with plant maturity (i.e., seedlings or sprouts vs. adult leaves as opposed to leaf maturity) may be a common pattern among conifers like S. sempervirens or P. taeda and distinct from non-conifers, like E. oleracea. Furthermore, a decrease in diversity of symbiotic fungal species with plant maturity has also been found in mycorrhizal associations as well as 16

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rhizosphere communities (Houlden et al. 2008; Husband et al. 2002a; Husband et al. 2002b). Given our findings along with similar correlations in other symbiotic systems, symbiont

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specialization associated with plant maturity may be common.

Culturing vs. environmental PCR

Cloning did not identify more distinct 99% ITS2 OTUs than culturing per sampling effort

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for both host ages (Table 1). In adult communities, cloning only identified two new OTUs, both of which were singletons (Fig. 3), and did not suggest that we were missing any significant

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fungal lineages with culturing methods (Suppl. Fig. 2). In contrast, cloning identified 26 new OTUs (3 new classes) in seedling communities, of which 7 were non-singletons (Fig. 1 & 3, Table 2). Some non-singleton OTUs were also from lineages uniquely detected with cloning, such as Stereum spp. in the Agaricomycetes (e52ss004 and e50ss011), Malassezia spp. in the

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Exobasidiomycetes (e50ss004), and a Leotiomycetes OTU with high ITS similarity to Monilina sp. (e50ss036). Some OTUs unique to cloning, such as e50ss015 (Penicillium sp.), have been found as cultures in other studies (Sandberg et al. 2014; Vega et al. 2010), suggesting that

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factors other than the ability to grow on MEA, such as competing endophytic species, might affect an endophyte’s ability to be cultured. Lastly, OTUs detected only with cloning may

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represent non-endophytic fungi from inefficient surface-sterilization. For example, Malassezia spp. are common fungi found on the human skin (Findley et al. 2013) and Peltigera spp. (e50ss002 and e50ss005) are lichens with a widespread distribution (Martinez et al. 2003). Arnold et al. (2007) also found Malassezia spp. and unexpected lichen species from cloning.

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OTU cas039 made up the majority of the clones, but not cultures, from seedling samples, despite being one of the most culturable OTUs from adult needles. We think the particular seedling samples we used for the bulk DNA extraction had an unusually high infection rate by

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this fungal species or there is a primer-binding bias. We found that different annealing

temperatures did not significantly affect the proportion of cas039 clones (data not shown). In the future, different pairs of primers may be a better way to overcome primer-bias.

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We found relatively few isolates belonging to the genus Lophodermium (Helotiales, Leotiomycetes), which was the most commonly cultured fungus in Arnold et al. (2007)’s study.

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Even common endophyte OTUs may have interannual variations that were not detected in this study, although sampling was conducted over two different seasons and no significant differences were detected (Fig. 2). Alternatively, Lophodermium endophytes may have been dominant in Arnold et al. (2007)’s study due to their comparatively fast growth rates, which

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increases their likelihood of culturing, especially from larger (> 2 mm) leaf sections. This is further supported by Arnold et al. (2007) finding considerably lower frequency of

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Lophodermium endophytes with cloning than expected based on their culturing results.

A diffuse mutualism with key players?

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Despite the increasing number of studies of endophytic fungi within multiple host species

and environments (Carroll 1988a; Ganley et al. 2004; Rodriguez et al. 2009; Sun et al. 2012; U'Ren et al. 2010; Zimmerman & Vitousek 2012), their ecological roles and factors underlying the community diversity remain mostly unknown (Rodriguez et al. 2009). Evidence supporting horizontally-transmitted endophytic fungi as mutualists are rare and circumstantial (Faeth & 18

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Fagan 2002; Sieber 2007 but see Carroll 1988; Arnold et al. 2003, Ganley et al. 2008) and have been under great speculation (Faeth 2002; Sieber 2007), mainly due to their low host specificity and their high phylogenetic diversity within hosts (Herre et al. 1999). Fungal endophyte

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communities within plants may be an example of a diffuse mutualistic network where the

benefits are low for the individual host, but are high for the host population (Carroll 1988a;

Merckx & Bidartondo 2008; Stanton 2003). Furthermore, the mutualism may be “unevenly

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diffuse”, where one or a few fungal species are particularly important on a specific host

individual despite the high diversity and high number of fungal partner species throughout the

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geographic range of the host species (Gove et al. 2007; Jordano 1987; Ridout and Newcombe 2015).

We found that some fungal endophytes are consistently more common than others at the adult stage, which suggests that these fungal endophytes are either better adapted to adult tissues,

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preferentially selected by adult tissues, their spores are disproportionately abundant in the canopy compared to the ground level, or a combination of these factors. Such species may have ecological roles that are distinct from others and should be further investigated. In pine needles,

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seedlings and adults alike contain various secondary metabolites, including phenolics, flavonoids, condensed tannins, and monoterpenes (Kainulainen et al. 1996) and their relative

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abundances with age depend on the particular compound (Oleszek et al. 2002; Thoss et al. 2007), which may affect the community structure of endophytes. They also vary in their vulnerability to different stress factors (Boege & Marquis 2005) as well as exposure to different fungal species even when they occur in close spatial proximity, which may lead to differential selection of endophytic fungi with different beneficial functions. However, host-specific species 19

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may still remain rare in the community despite any beneficial roles due to other ecological factors (Wilson & Yoshimura 1994; Reveillaud et al. 2014), e.g., poor survival outside of host or competition with other endophytes. The future challenge will be to identify functional

factors affect the diversity of endophytes within individual hosts.

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ACKNOWLEDGEMENTS

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differences among common and rare endophytic fungal species as well as understanding what

We thank A. E. Arnold and the anonymous reviewers for thoughtful comments on a draft. We

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thank S. Jiang and A. Gartin, and P. Manos for their cooperation or assistance in lab- and field work. This research was supported by the Molecular Mycological Pathogenesis Training Program (MMPTP, Duke University) to RO and grant NSF DEB-1046065 to FL. This work was performed in the Duke Forest, Duke University’s outdoor teaching and research laboratory at the

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site of the former FACE facility, which was operated in partnership through the Brookhaven National Laboratory and Duke University. The authors have no conflict of interest to declare.

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REFERENCES

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Arnold AE & Herre EA (2003) Canopy cover and leaf age affect colonization by tropical fungal endophytes: Ecological pattern and process in Theobroma cacao (Malvaceae). Mycologia 95: 388-398. Arnold AE & Lutzoni F (2007) Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology 88: 541-549. Arnold AE, Henk DA, Eells RL, Lutzoni F & Vilgalys R (2007) Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR. Mycologia 99: 185-206.

20

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Arnold AE, Mejia LC, Kyllo D, Rojas EI, Maynard Z, Robbins N & Herre EA (2003) Fungal endophytes limit pathogen damage in a tropical tree. Proceedings of the National Academy of Sciences USA 100: 15649-15654.

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Bae H, Sicher RC, Kim MS, Kim S-H, Strem MD, Melnick RL & Bailey BA (2009) The beneficial endophyte Trichoderma hamatum isolate DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao. Journal of Experimental Botany 60: 32793295. Bazzicalupo AL, Balint M & Schmitt I (2013) Comparison of ITS1 and ITS2 rDNA in 454 sequencing of hyperdiverse fungal communities. Fungal Ecology 6: 102.

SC

Boege K & Marquis RJ (2005) Facing herbivory as you grow up: the ontogeny of resistance in plants. Trends in Ecology & Evolution 20: 441-448.

M AN U

Bormann FH (1956) Ecological implications of changes in the photosynthetic response of Pinus taeda seedlings during ontogeny. Ecology 37: 70-75. Cannone JJ, Subramanian S, Schnare MN, Collett JR, R’Souza LM, Du Y, Feng B, Lin N, Madabusi LV, Müller KM, Pande N, Shang Z, Yu N, Guttell RR (2002) The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics 3:15.

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Caporaso JG, Kuczynski J, Stombaugh J, et al. (2010) QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7: 335-336. Carroll G (1988) Fungal endophytes in stems and leaves - from latent pathogen to mutualistic symbiont. Ecology 69: 2-9.

EP

Carroll GC & Carroll FE (1978) Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Canadian Journal of Botany-Revue Canadienne de Botanique 56: 3034-3043.

AC C

Colwell RK (2013) EstimateS: Statistical estimation of species richness and shared species for samples. Vol. Version 9 purl.oclc.org/estimates. Diamandis S (1981) A serious attack on Pinus brutia L. in Greece. Current research on conifer needle diseases,(Millar CS, ed.) pp. 9-12. Aberdeen University Press, Aberdeen, Scotland. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460-2461. Espinosa Garcia FJ & Langenheim JH (1990) The endophytic fungal community in leaves of coastal redwood population - diversity and spatial patterns. New Phytologist 116: 89-97. 21

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Faeth SH (2002) Are endophytic fungi defensive plant mutualists? Oikos 98: 25-36. Faeth SH & Fagan WF (2002) Fungal endophytes: Common host plant symbionts but uncommon mutualists. Integrative and Comparative Biology 42: 360-368.

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Ferreira Rodrigues K (1994) The foliar fungal endophytes of the Amazonian palm Euterpe oleracea. Mycologia 86: 376-385.

Findley K, Oh J, Yang J, et al. (2013) Topographic diversity of fungal and bacterial communities in human skin. Nature 498: 367-370.

SC

Frohlich J, Hyde KD & Petrini O (2000) Endophytic fungi associated with palms. Mycological Research 104: 1202-1212.

M AN U

Ganley RJ, Brunsfeld SJ & Newcombe G (2004) A community of unknown, endophytic fungi in western white pine. Proceedings of the National Academy of Sciences of the United States of America 101: 10107-10112. Ganley RJ, Sniezko RA & Newcombe G (2008) Endophyte-mediated resistance against white pine blister rust in Pinus monticola. Forest Ecology and Management 255: 2751-2760.

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Gardes M & Bruns TD (1993) ITS primers with enhanced specificity for Basidiomycetes Application to the identification of mycorrhizae and rusts. Molecular ecology 2: 113-118. Gove AD, Majer JD & Dunn RR (2007) A keystone ant species promotes seed dispersal in a "diffuse" mutualism. Oecologia 153: 687-697.

EP

Hendrey GR, Ellsworth DS, Lewin KF & Nagy J (1999) A free-air enrichment system for exposing tall forest vegetation to elevated atmospheric CO2. Global Change Biology 5: 293-309.

AC C

Herre EA, Knowlton N, Mueller UG & Rehner SA (1999) The evolution of mutualisms: exploring the paths between conflict and cooperation. Trends in Ecology and Evolution 14: 4953. Houlden A, Timms-Wilson TM, Day MJ & Bailey MJ (2008) Influence of plant developmental stage on microbial community structure and activity in the rhizosphere of three field crops. FEMS Microbiology Ecology 65: 193-201. Husband R, Herre EA & Young JPW (2002) Temporal variation in the arbuscular mycorrhizal communities colonising seedlings in a tropical forest. FEMS Microbiology Ecology 42: 131-136.

22

ACCEPTED MANUSCRIPT

Husband R, Herre EA, Turner SL, Gallery R & Young JPW (2002) Molecular diversity of arbuscular mycorrhizal fungi and patterns of host association over time and space in a tropical forest. Molecular Ecology 11: 2669-2678.

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Jordano P (1987) Patterns of mutualistic interactions in pollination and seed dispersal connectance, dependence asymmetries, and coevolution. American Naturalist 129: 657-677. Kainulainen P, Holopainen J, Palomaki V & Holopainen T (1996) Effects of nitrogen fertilization on secondary chemistry and ectomycorrhizal state of Scots pine seedlings and on growth of grey pine aphid. Journal of Chemical Ecology 22: 617-636.

SC

Kumaresan V & Suryanarayanan TS (2002) Endophyte assemblages in young, mature and senescent leaves of Rhizophora apiculata: evidence for the role of endophytes in mangrove litter degradation. Fungal Diversity 9: 81-91.

M AN U

Lozupone C, Hamady M & Knight R (2006) UniFrac - An online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7: 371. Luecking R, Hodkinson BP, Stamatakis A & Cartwright RA (2011) PICS-Ord: unlimited coding of ambiguous regions by pairwise identity and cost scores ordination. BMC Bioinformatics 12: 10.

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Machouart M, Samerpitak K, de Hoog GS & Gueidan C (2014) A multigene phylogeny reveals that Ochroconis belongs to the family Sympoventuriaceae (Venturiales, Dothideomycetes). Fungal Diversity 65: 77-88. Maddison DR & Maddison WP (2005) MacClade 4: Analysis of phylogeny and character evolution. http://macclade.org.

EP

Martinez I, Burgaz AR, Vitikainen O & Escudero A (2003) Distribution patterns in the genus Peltigera Willd. Lichenologist 35: 301-323.

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Merckx V & Bidartondo MI (2008) Breakdown and delayed cospeciation in the arbuscular mycorrhizal mutualism. Proceedings of the Royal Society B 275: 1029-1035. Miller JD, Sumarah MW & Adams GW (2008) Effect of a rugulosin-producing endophyte in Picea glauca on Choristoneura fumiferana. Journal of chemical ecology 34, 362-368. Minter DW (1981) Possible biological control of Lophodermium seditiosum. Current research on conifer needle diseases (Millar CS, ed.) p. 67-74. Aberdeen University Press, Aberdeen, Scotland.

23

ACCEPTED MANUSCRIPT

Moncalvo JM, Lutzoni FM, Rehner SA, Johnson J & Vilgalys R (2000) Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Systematic Biology 49: 278-305.

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Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N & Larsson K-H (2008) Intraspecific ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evolutionary Bioinformatics 4: 193-201. Oleszek W, Stochmal A, Karolewski P, Simonet AM, Macias FA & Tava A (2002) Flavonoids from Pinus sylvestris needles and their variation in trees of different origin grown for nearly a century at the same area. Biochemical Systematics and Ecology 30: 1011-1022.

SC

Pitkaranta M, Meklin T, Hyvärinen A, Paulin L, Auvinen P, Nevalainen A & Rintala H (2008) Analysis of fungal flora in indoor dust by ribosomal DNA sequence analysis, quantitative PCR, and culture. Applied Environmental Microbiology 74: 233-244.

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Price EW & Carbone I (2005) SNAP: workbench management tool for evolutionary population genetic analysis. Bioinformatics 21: 402. Prieto M, Baloch E, Tehler A & Wedin M (2013) Mazaedium evolution in the Ascomycota (Fungi) and the classification of mazaediate groups of formerly unclear relationship. Cladistics 29: 296-308.

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Quaedvlieg W, Verkley GJM, Shin HD, Barreto RW, Alfenas AC, Swart WJ, Groenewald JZ & Crous PW (2013) Sizing up Septoria. Studies in Mycology 307-390. Redman RS, Dunigan DD & Rodriguez RJ (2001) Fungal symbiosis from mutualism to parasitism: who controls the outcome, host or invader? New Phytologist 151: 705-716.

EP

Reveillaud J, Maignien L, Eren AM, Huber JA, Apprill A, Sogin ML, Vanreusel A (2014) Hostspecificity among abundant and rare taxa in the sponge microbiome. ISME Journal 8: 11981209.

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Ridout M & Newcombe G (2015) The frequency of modification of Dothistroma pine needle blight severity by fungi within the native range. Forest Ecology and Management 337, 153-160. Rodriguez RJ, White JF, Arnold AE & Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytologist 182: 314-330. Saikkonen K, Faeth SH, Helander M & Sullivan TJ (1998) Fungal endophytes: a continuum of interactions with host plants. Annual Review of Ecology, Evolution, and Systematics 29: 319-343.

24

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Sandberg DC, Battista LJ & Arnold AE (2014) Fungal endophytes of aquatic macrophytes: diverse host-generalists characterized by tissue preferences and geographic structure. Microbial Ecology 67: 735-747.

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Schmidt P-A, Balint M, Greshake B, Bandow C, Roembke J & Schmitt I (2013) Illumina metabarcoding of a soil fungal community. Soil Biology & Biochemistry 65: 128-132. Schoch CL, Crous PW, Groenewald JZ, et al. (2009) A class-wide phylogenetic assessment of Dothideomycetes. Studies in mycology 64: 1-15.

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Schoch CL & Seifert KA & Huhndorf S, et al. (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America 109: 6241-6246. Schulz B & Boyle C (2005) The endophytic continuum. Mycological Research 109: 661-686.

M AN U

Sieber TN (2007) Endophytic fungi in forest trees: are they mutualists? Fungal Biology Reviews 21: 75-89. Spatafora JW, Sung G-H, Johnson D, et al. (2006) A five-gene phylogeny of Pezizomycotina. Mycologia 98: 1018-1028.

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Stamatakis A (2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688. Stamatakis A, Hoover P & Rougemont J (2008) A rapid bootstrap algorithm for the RAxML Web Servers. Systematic Biology 57: 758-771.

EP

Stanton ML (2003) Interacting guilds: moving beyond the pairwise perspective on mutualisms. American Naturalist 162: S10-23.

AC C

Thoss V, O'Reilly-Wapstra J & Iason GR (2007) Assessment and implications of intraspecific and phenological variability in monoterpenes of Scots pine (Pinus sylvestris) foliage. Journal of Chemical Ecology 33: 477-491. U'Ren JM, Lutzoni F, Miadlikowska J & Arnold AE (2010) Community analysis reveals close affinities between endophytic and endolichenic fungi in mosses and lichens. Microbial Ecology 60: 340-353. U'Ren JM, Dalling JW, Gallery RE, Maddison DR, Davis EC, Gibson CM & Arnold AE (2009) Diversity and evolutionary origins of fungi associated with seeds of a neotropical pioneer tree: a case study for analysing fungal environmental samples. Mycological Research 113: 432-449.

25

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Vega FE, Simpkins A, Aime MC, Posada F, Peterson SW, Rehner SA, Infante F, Castillo A & Arnold AE (2010) Fungal endophyte diversity in coffee plants from Colombia, Hawai'i, Mexico and Puerto Rico. Fungal Ecology 3: 122-138.

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Vilgalys R & Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238-4246. Wang Z, Johnston PR, Takamatsu S, Spatafora JW & Hibbett DS (2006) Toward a phylogenetic classification of the Leotiomycetes based on rDNA data. Mycologia 98: 1065-1075.

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Wilson DS & Yoshimura J (1994) On the coexistence of specialists and generalists. American Naturalist 144: 692-707.

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Zimmerman NB & Vitousek PM (2012) Fungal endophyte communities reflect environmental structuring across a Hawaiian landscape. Proceedings of the National Academy of Sciences of the United States of America 109: 13022-13027.

FIGURE CAPTIONS

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Fig. 1 Venn diagram of endophytic fungi (OTUs = 99% ITS2 similarity groups) recovered from needle tissues of seedlings and adult trees of P. taeda using culturing and cloning methods. The number and percentage of overlapping, non-overlapping non-singleton, and non-overlapping singleton OTUs are represented in the circles. Outer circles represent singletons, OTUs that are found only once in the comparison. Inner circles represent non-singletons.

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Fig. 2 Comparison of fungal endophyte communities cultured from needles of seedlings and adult trees of P. taeda sampled in the summer and winter. a) Principal coordinates analysis of fungal endophyte communities with weighted abundance without singletons using the UniFrac metric. Dotted circles indicate >99% jackknife support clusters after 1000 permutations. b) Hierarchical clustering of communities with unweighted abundance without singletons using UPGMA based on tree represented in Fig. 3. Jackknife support value (1000 permutations) is shown for the edge separating the two clusters. Scale bar shows UniFrac distance. Open blue circles represent fungal endophyte communities in seedling needles and closed green circles represent those in needles of adult trees. Light blue or green circles indicate communities from summer samples and the dark blue and green circles indicate communities from winter samples. Fig. 3 Maximum likelihood (ML) phylogenetic tree based on 312 LSU rRNA sequences showing the phylogenetic placement of 117 fungal endophyte OTUs found in needles of P. taeda seedlings and adult trees as part of this study (shown in bold) within the broader context of the dikarya represented by 149 reference species and 46 genotypes from Arnold et al. (2007). Phylogenetic searches were conducted in three ways: i) 292 unambiguously aligned sites only, ii) 292 unambiguously aligned sites with 162 ambiguous sites recoded with PICS-Ord, and iii) 292 26

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unambiguously aligned sites with 162 ambiguous sites recoded with PICS-Ord and with conservative backbone constraint tree (Suppl. Fig. 1). The tree shown here is derived from analysis type iii. The number of isolates or clones for each OTU (99% ITS2) in each sample is represented by the size of the circles on the right. The percentage of isolates or clones for OTUs that made up more than 10% of the sample is indicated within the circles. Clones are from pine needle tissue collected from summer. The scale bar indicates the number of substitutions per site for a unit branch length. Branches were bolded if ML bootstrap values were >70% for all three types of analyses. When branches were only highly supported by analysis types ii and iii, the ML bootstrap values of the analysis type ii were indicated near branches. Nodes for major fungal classes are indicated with a diamond. Sequences from Arnold et al. (2007) are represented by grey code names (e.g., 2235, c0280) and starts with “c” if it was a clone.

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Fig. 4 Rarefaction curves of endophytic fungal communities from needle tissues of P. taeda seedlings and adult trees. Curves include black line representing observed 99% OTUs, shaded area around curve representing the 95% confidence intervals, and extrapolated curve up to 2x the number of isolates from the seedling community. Dotted lines represent bootstrap species richness. Communities only include cultured isolates. Rarefaction curves of clones and cultures using 95% and 99% ITS2 similiarity are reported in Suppl. Fig. 2.

27

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TABLES Unidirectionally sequenced

Identified by RFLP match

50 (51%)

36 (72%)

14 (28%)

0

57 (69%)

57 (100%)

0

N/A

96

28 (29%)

142/204 (69.6%) 171/204 (83.8%)

97 (68%)

32 (33%)

95 (55%)

27 (28%)

N/A

96

2 (2%)

SC 0

25 (26%)

43 (45%)

45 (46%)

20 (21%)

49 (52%)

19 (20%)

6 (6%)

88 (92%)

34 (28.8%) 18 (15.3%) 25 (21.2%) 6 (5.1%)

170 118 (34.6%) Table 1. Sampling summary and genotyping methods of endophytic fungi from needle tissues of seedling and adult P. taeda from summer and winter seasons. Isolation frequencies indicate fractions from which fungal cultures were isolated from the total of 2 mm needle segments on MEA media. Genotyped culture fractions indicate those genotyped out of the total number of cultured isolates. Cultures were either genotyped by bidirectional sequencing, unidirectional sequencing, or RFLP matching. Percent OTUs indicate OTUs out of a total of 118 OTUs delimited using the conservative 99% similarity criterion. 491

182 (56.7%)

321 (65.4%)

EP

N/A

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Total

2 3 4 5 6

98/564 (17.4%) 82/720 (11.4%)

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Summer seedling Winter seedling Summer seedling clone Summer adult Winter adult Summer adult clone

99% ITS2 OTUs 35 (29.7%) 37 (31.4%)

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Bidirectionally sequenced

Isolation Genotyped frequency

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1

1

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7 % genotyped isolate

% clone

EP

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8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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Taxonomic group/OTU Seedling Adult Seedling Adult Ascomycota 98.1 69.3 83.3 88.5 Sordariomycetes 12.5 2.1 62.6 30.7 1 csw046 9.3 0.01 0.01 0 csw043 7.52 0.01 5.2 0 csw026 4.53 0 2.1 0 3 css003 4.5 0 0.9 0 csw042 1.9 7.83 2.1 0 Leotiomycetes 7.5 8.3 4.2 41.7 caw049 0 3.6 0 41.72 Dothideomycetes 26.2 29.2 53.1 44.8 cas039 0.9 26.62 34.41 43.71 csw009 2.8 0 8.33 0 Lecanoromycetes 0 0 2.1 0 Eurotiomycetes 1.9 0.5 13.5 0 e50ss015 0 0 9.42 0 Pezizomycetes 0 0.5 0 0 Basidiomycota 1.9 30.7 15.6 11.5 Tremellomycetes 0 0 2.1 0 Agaricomycetes 0.9 0 11.5 0 Atractiellomycetes 0.9 30.7 0 11.5 caw010 0.9 30.71 0 11.53 Exobasidiomycetes 0 0 2.1 0 Unknown 0 0 1.0 0 Table 2. Taxonomic distribution of fungal endophytes in needles of seedling and adult P. taeda detected with culturing and cloning methods. Summer and winter samples are pooled for culturing. The three most commonly found OTUs from each sampling type are indicated with superscripts 1, 2, and 3. The class with the most OTUs from each sampling type is bolded.

2

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27 % in seedling

Wilcoxon rank test

caw010

31.1 ± 15.4

0.6 ± 1.4

p < 0.05

cas039

25.3 ± 15.2

1.1 ± 2.7

p < 0.05

csw042

8.1 ± 10.2

1.2 ± 2.8

p = 0.18

csw046

3.3 ± 5.8

8.1 ± 6.0

p = 0.28

csw043

2.4 ± 5.8

6.9 ± 6.2

p = 0.42

css003

0.0 ± 0.0

5.3 ± 6.6

p = 0.18

csw026

0.0 ± 0.0

4.0 ± 6.2

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% in adult

p = 0.37

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Table 3. Most commonly cultured OTUs from adult and seedling tissues (Table 2). Average frequency and standard deviation of each OTU per plot per season are indicated for adult trees and seedlings. Results of the Wilcoxon signed-rank test to determine if commonly cultured endophytes were found in needles of adult trees or seedlings more often than by chance.

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28 29 30 31 32 33 34 35 36 37 38

OTU

3

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CULTURES

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17

(6.7%) (18.9%)

16

(43.6%) (41.0%)

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(17.8%)

17

6

CULTURES & CLONES Seedlings Adult Trees

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(46.7%) (10.0%)

16

62

Adult Trees

Seedlings

EP

9

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42

Adult Trees

SC

Seedlings

CLONES

15

(52.5%) (12.7%)

17 (14.4%)

6

18

(5.1%) (15.2%)

1 (2.6%)

3 (7.7%)

2 (5.1%)

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b

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0.4

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SC

0.2 0

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-0.2

99%

EP

99%

-0.4

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P2 - Percent variation explained: 21.9%

a

-0.6 -0.6

-0.4

-0.2

0

0.2

0.05

0.4

0.6

P1 - Percent variation explained: 61.2%

0.1

Seed ling summ Seed er ling winte r Adult summ er Adult winte r Seed ling clone Adult clone

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Thelephora vialis

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Myxarium mesonucleatum e52ss039 Myxarium sp. Asterodon ferruginosa Phellinus wahlbergii c0256 Phellinus gilvus Acantholichen pannarioides Vuilleminia cystidiata e50ss041 Dentocorticium sulphurellum Perenniporia japonica Phlebia acerina 86   Antrodia albida e50ss008 csw002 Peniophora incarnata Peniophora nuda 100   e52ss004 Stereum hirsutum 100   e50ss011 Stereum rugosum Athelopsis subinoconspicua 100   Leptosporomyces raunkiageri e50ss027 e52ss029 Cryptococcus sp. Cryptococcusl arabidensis Cryptococcus sp.Cryptococcus arabidensis e52ss020 99   e52ss020 e52ss002 e52ss002 Ustilago sparsa Ustilago nuda Ustilago nuda Ustilago sparsa Ustilago cynodontis c0264 Malassezia dermatis Malassezia dermatis e50ss004 e50ss004 c0263 Malassezia restricta Malassezia restricta c0263 Rhodotorula pallida Naohidea sebacea Sporobolomyces Sporobolomycesfoliicola foliicola Rhodotorula pallida Naohidea sebacea Microbotryum reticulatum Melampsora lini Zymoxenogloea eriophori Helicobasidium purpureum c0248 Septobasidium sp. Rhodotorula hordea eriophori Zymoxenogloea Ustilentyloma fluitans reticulatum Microbotryum Helicogloea variabilis c0248 Melampsora lini Rhodotorula hordea Helicobasidium purpureum Ustilentyloma fluitans Septobasidium sp. Platygloea vestita SaccoblastiaPlatygloea farinacea vestita Phleogena Phleogenafaginea faginea caw010 Saccoblastia farinacea caw010 2617 96   2027A 2027A 2617 e50ss008 Neolecta vitellina csw002 Schizosaccharomyces pombe Peniophora incarnata

97  

SC

86  

95  

84  

(to Ascomycota)

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AC C

EP

     

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100   82  

Agaricomycetes

Tremellomycetes

Ustilaginomycetes

Exobasidiomycetes Cystobasidiomycetes Pucciniomycetes Microbotryomycetes 11%# 16%#

Atractiellomycetes 18%#

38%   23%#

11%  

(to rest of Dikarya)

Taphrinomycotina Saccharomycotina Pezizomycetes Orbiliomycetes

77$

Coniocybomycetes

Leotiomycetes

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Lichinomycetes

Seed ling summ Seed er ling winte r Adult summ er Adult winte r Seed ling clone Adult clone

Seed ling summ Seed er ling winte r Adult summ er Adult winte r Seed ling clone Adult clone

css041 c0245 csw009 c0251 cas074brachyspora Curvularia Cladosporium sphaerospermum css038 e50ss021 Alternaria alternata c0260 Alternaria limoniasperae css037 c0280 c0261 c0249 c0250e52ss045 1951 e52ss010 1894 Leotia lubrica Collophora africana c0246 1990 95$ Hortaea werneckii css043 Mycosphaerella molleriana Flagellospora curvula csw006 Collophora paarla Mycosphaerella marksii Lophodermium pinastri csw013 2185 e50ss009csw019 76$ e50as039cas073 csw014 c0258 css044 c0265 csw021 e50ss029 1815 css039 1757 csw007 Lophodermium conigenum 96$ Cercospora zebrina e50ss036 Leotia lubrica Monilinia laxa 100$ css043 Sclerotium cepivorum Collophora africana Cudoniella clavus 96$ 1990 Hymenoscyphus ericae 82$ Collophora Fabrella tsugae paarla Flagellospora curvula 100$ cas080 Lophodermium pinastri csw024 75$ 2247 1815 caw049 2185 e50ss009 76$ c0268 1734 e50as039 csw025 c0258 Xylomelasma sordida css044 csw031 csw021 Hypocrea schweinitzii 87$ 1757 Fusarium lichenicola Lophodermium conigenum csw054 e50ss036diatrypelloidea Cytospora Monilinia csw053 laxa Sclerotium Taeniolella altacepivorum Cudoniella csw039 clavus Tubakia dryina Hymenoscyphus ericae caw069 Fabrella tsugae 100$ 1981A cas080 csw024 Lasiosphaeria sorbina

ACCEPTED MANUSCRIPT

Eurotiomycetes

42%$

SC

Geoglossomycetes Lecanoromycetes

css050 2247 caw093 csw025 Lasiosphaeris hirsuta caw049 Neurospora crassa c0268 Sordaria macrospora 95$ 1734 appendiculata Podospora csw026Glomerella cingulata 2450 Paramicrothyrium chinensis Colletotrichum gloeosporioides css033 Glomerella cingulata 100$ 1814 2450 css034 css034 Hypocrea schweinitzii 1814 csw054 Colletotrichum gloeosporioides Fusarium lichenicola csw052 csw053 Coniochaeta prunicola 92$ Cytospora diatrypelloidea csw036 Taeniolella alta Lecythophora mutabilis 83$ 2350 csw039 100$ Tubakia dryina 2100 1981A eucalypti Pseudophloeospora 100$ cas088caw069 Virgaria nigra Paramicrothyrium chinensis 75$ css032 css033 2425 csw052 Hypoxylon crocopeplum csw036 Hypoxylon haematostroma Coniochaeta prunicola 100$ csw032 Lecythophora mutabilis 98$ caw091 2100 95$100$ 2204 2350 cas091 Neurospora crassa css025 Sordaria macrospora 81$ css024 caw055 Podospora appendiculata csw026nitens Annulohypoxylon css050 100$ caw051 DaldiniaLasiosphaeria eschscholzii sorbina css023 Lasiosphaeris hirsuta caw059 caw093 caw042 Xylomelasma sordida csw057 csw031 e52ss016 Pseudophloeospora eucalypti css029 100$ cas088 Pseumassariella vexata Virgaria nigra 88$ 1817 css032 Biscogniauxia mediterranea 2425 2151 Hypoxylon crocopeplum csw035 css022 Hypoxylon haematostroma 90$ Xylariacsw032 hypoxylon 90$ caw091 css030 cas097 cas091 css019 2204 css020 css025 csw046 css024 csw051 caw055 Anthostomella eucalyptorum Annulohypoxylon nitens Muscodor fengyangensis caw054 Daldinia eschscholzii 97$ cas089 caw051 99$ Muscodor albuscss023 caw059 cas090 100$ 2281 caw042 cas096 csw057 Xylaria coccophora Anthostomella eucalyptorum 2207 csw033 Nemania bipallata css031 100$ Nemania diffusa sphaerica Nigrospora 99$ 2235 csw043 100$ e50as008 95$ css003 csw042 Nigrospora oryzae css026 caw052 caw060 css006 css028 Xylaria badia caw090 78$ css027 Xylaria badia csw055 csw055 100$ Coniolariella hispanica Coniolariella hispanica caw090 css031 98$ css028 csw033 css026 100$ Nigrospora sphaerica caw060 csw043 12%$ Pseumassariella vexata css003 10%$ css029 78$ Nigrospora oryzae 2151 caw052 99$ 99$ csw035 css006 Biscogniauxia mediterranea 1817 2207 cas096 Xylaria coccophora

M AN U

Xylonomycetes

Arthoniomycetes

11%# 16%#

35%$

18%#

34%$ 44%$

TE D

23%#

EP

AC C

Pezizomycotina

(to

Helicogloea variabilis e52ss039 Myxarium sp. Melampsora lini Thelephora vialis Helicobasidium purpureum Phlebia acerina Septobasidium sp. Antrodia albida Vuilleminia cystidiata Platygloea vestita Perenniporia japonica Phleogena faginea e50ss041 Dentocorticium sulphurellum Saccoblastia farinacea Cryptococcus sp. caw010 Basidiomycota, Fig. 2a) Cryptococcus arabidensis 2027A e52ss020 e52ss002 2617 Ustilago sparsa Neolecta vitellina Ustilago nuda Schizosaccharomyces pombe Ustilago cynodontis c0264 Candida albicans Malassezia dermatis Saccharomyces cerevisiae e50ss004 Morchella esculenta Malassezia restricta 94$ c0263 Urnula hiemalis Naohidea sebacea cas086 Sporobolomyces foliicola Orbilia vinosa Rhodotorula pallida Melampsora lini Orbilia auricolor Helicobasidium purpureum e50ss030 Septobasidium sp. Sclerophora coniophaea Zymoxenogloea eriophori Microbotryum reticulatum brachypoda Chaenotheca c0248 Lempholemma polyanthes Rhodotorula hordea Peltula umbilicata 88$ Ustilentyloma fluitans Platygloea vestita Peltula obscurans Saccoblastia farinacea Caliciopsis orientalis Phleogena faginea 80$ Penicillium variabile caw010 2617e52ss006 95$ 2027A Aspergillus fumigatus 89$ Neolecta vitellina e52ss043Schizosaccharomyces pombe Penicillium freii Candida albicans 98$ Penicillium chrysogenum Saccharomyces cerevisiae Morchella esculenta 99$ e50ss015 cas086 Urnula hiemalis Capronia pilosella Orbilia vinosa Exophiala dermatitidis Orbilia auricolor 92$ Pyrenula pseudobufonia 98$ e50ss030 Caliciopsis orientalis Rhynchostoma proteae e52ss006 77$ 2072 Penicillium variabile csw020 Aspergillus fumigatus 96$ e52ss043 Phaeomoniella effusa Penicillium freii Phaeoacremonium chlamydosporum e50ss015 css049 Penicillium chrysogenum 80$ Exophiala dermatitidiscapensis Phaeomoniella Capronia pilosella Trichoglossum hirsutum Pyrenula pseudobufonia Geoglossum barlae Rhynchostoma proteae Phaeoacremonium Pleopsidium gobiense chlamydosporum css049 Teloschistes exilis Phaeomoniella capensis 76$ Usnea antarctica Phaeomoniella effusa csw020 Diabaeis baeomyces 2072 e50ss002 coniophaea Sclerophora Peltigera monticola Chaenotheca brachypoda Peltigera degenii Lempholemma polyanthes 91$ Peltula umbilicata Peltigera canina Peltula obscurans Xylonoma hevea Xylona heveae Trichoglossum hirsutum Phaeococcomyces nigricans Geoglossum barlae 94$ c0285 c0285 Pleopsidium gobiense 90$ Lecanographa Lecanographa dimleaenoides Teloschistes exilis UsneaDendrographa antarctica decolorans Dendrographa decolorans Diabaeis baeomyces Dendrographa leucophaea minor Dendrographa e50ss002 Venturia populina Peltigera monticola Venturia populina Peltigeracordae degenii Fusicladium Fusicladium cordae Peltigera canina caw040 Myriangium caw040 duriaei Ochroconisanellii anellii 89$ 2448 Ochroconis cas038 Ochroconis sp. 2 99$ PhaeococcomycesOchroconis nigricans sp. 2 Ochroconis sp. 1 c0285 Ochroconis sp. 1 Lecanographa css047dimleaenoides css047 Dendrographa decolorans c0272 100$ Dendrographa leucophaea c0272 Septorioides pini−thunbergii Venturia populina Septorioides pini−thunbergii Fusicladium cordae 100$ cas039 cas039 caw040 91$ 2092 Ochroconis anellii 2092 Botryosphaeria ribis 95$ Ochroconis sp. 2 css046Ochroconis sp. 1 css046 Botryosphaeria css047ribis css045 c0272 Guignardia css045 mangiferae Septorioides pini−thunbergii 1855 100$ 1855 cas039 2092 GuignardiaMyriangium mangiferae duriaei css046 cas038 csw004 100$ Botryosphaeria ribis 2448 globosum css045 Anteaglonium 1855 2324 css048 mangiferae Guignardia csw005 96$ 100$ e52ss046 csw004 Anteaglonium globosum Dothidea ribesia Letendraea helminthicola css048 e52ss036 84$ e52ss046 c0245 2294 Letendraea helminthicola c0251 Aureobasidium pullulans c0245 Curvularia c0251 brachyspora e50ss019 Curvularia css038 brachyspora Capnodium citri css038 css037 89$ css037 css041 Alternaria alternata Alternaria alternata csw009 Alternaria limoniasperae Alternaria limoniasperae 100$ cas074 c0249 c0249 Cladosporium sphaerospermum 1951 c0250 1951 92$ e50ss021 Trematosphaeria heterospora c0250 100$ c0280 Neosetophoma samarorum Trematosphaeria heterospora csw018 c0260 86$ 2231 Neosetophoma samarorum e52ss045 81$ css035 e52ss010 csw018 e50ss017 94$ Epicoccum c0261 nigrum 2231 Phoma bellidis Mycosphaerella molleriana 90$ css035 csw017 csw006 80$ csw015 e50ss017 csw016 Hortaea nigrum werneckii Epicoccum 79$ 2324 c0246 Phoma bellidis 91$ csw005 1894 Dothidea ribesia csw017 Mycosphaerella marksii e52ss036 csw015 2294 csw013 72$ Aureobasidium pullulans csw016 cas073 100$ e50ss019 2324 92$ Capnodium citri csw019 csw005 csw014 css041 csw009 Dothidea ribesia c0265 cas074 e52ss036 Cladosporium sphaerospermum e50ss029 e50ss021 2294 css039 c0260 Aureobasidium pullulans c0280 95$ 99$ csw007 e50ss019 c0261 Cercospora zebrina e52ss045 Capnodium citri Anteaglonium globosum e52ss010 csw004 css041 1894 c0246 css048 csw009 100$ Hortaea werneckii Letendraea helminthicola 100$ cas074 Mycosphaerella molleriana e52ss046sphaerospermum Cladosporium csw006 Mycosphaerella marksii Trematosphaeria heterospora e50ss021 csw013 2231 97$ c0260 csw019 csw018 c0280 cas073 csw014 Neosetophoma samarorum c0261 c0265 csw016e50ss029 91$ e52ss045 csw015 css039 97$ e52ss010 csw017 csw007 Cercospora zebrina 1894 css035 c0246 Leotia lubrica e50ss017 css043 Hortaea werneckii Collophora africana Phoma bellidis 1990 Mycosphaerella molleriana Epicoccum Collophora paarla nigrum csw006 c0245 Flagellospora curvula 72$ Lophodermium pinastri Mycosphaerella marksii c0251 85$ 1815 csw013 Curvularia brachyspora 2185 csw019 css038 88$ e50ss009 e50as039 Alternaria alternata cas073 c0258 91$ Alternaria limoniasperae csw014 95$css044 css037 csw021 c0265 c0249 e50ss029 84$ 1757 Lophodermium conigenum c0250 css039 e50ss036 (to Leotiomycetes & 1951Monilinia laxa csw007 SclerotiumLeotia cepivorum lubrica Cercospora zebrina clavus Sordariomycetes) Cudoniella Collophora africana ericae Hymenoscyphus Leotia lubrica 1990 Fabrella tsugae cas080 css043 css043 csw024Collophora africana

Dothideomycetes

Sordariomycetes

0.2

11%# 16%# 18%# 23%#

11%# 16%# 18%# 23%#

ACCEPTED MANUSCRIPT

RI PT SC

100

M AN U

TE D

Adult trees

EP

50

Seedlings

0

AC C

OTU

150

107

Isolates

192 214

ACCEPTED MANUSCRIPT

100%

97%

99% Septorioides pini-thunbergii

Accession

#

>99% ITS2

#

>99% ITS2

EF419944

#

>99% ITS2

KF251243

1 csw003

cas040-058, cas060, cas066, 34 cas068-072

3 cas073

100%

100% Dothistroma pini

KF251155

1

100%

100% Cladosporium sphaerospermumJX156365

1

5 cas080

100%

6 cas086

100%

99%

JQ761939

1

7 cas088

100%

100%

EF634381

1

8 cas089

100%

100%

JQ760849

1

9 cas090

100%

100%

JQ761880

94%

AM749940

1

JQ760905

1

99% Anthostomella sepelibilis

AY908989

1

100%

99%

AY969455

100%

99%

JQ760112

15 caw042

100%

99% Hypoxylon petriniae

JQ009309

16 caw049

100%

17 caw051

96%

18 caw052

100%

19 caw054

100%

20 caw055

100%

21 caw059

100%

22 caw060

95%

23 caw069

100%

24 caw090

100%

99% 97% Daldinia placentiformis 100% Nigrospora sphaerica 99% Muscodor yucatanensis 100% 99% Hypoxylon fragiforme 99% Nemania serpens 100% Dicarpella dryina 98% Xylaria frustulosa 100% Annulohypoxylon annulatum

1 csw001

JN198512

JN673055

1

HQ608152

css001-002, css0045 005

100% Nigrospora oryzae

EU529993

1

29 css019

2 caw092

100%

100% Anthostomella sepelibilis

AY908989

1

JN942179

1

30 css020

100%

99%

JQ760112

31 css022

100%

100%

KJ188097

2 css021

32 css023

100%

JQ009307

1

1

JQ761019

1

34 css025

93%

35 css026

100%

100% Nemania aenea

AJ390427

1

36 css027

100%

100% Xylaria oxyacanthae

GU322434

1

37 css028

100%

99% Xylaria frustulosa

JN673055

1

38 css029

100%

97% Pseudomassariella vexata

JF440977

1

39 css030

100%

88%

40 css031

95%

AY909002

1

1

1 1

42 css033

100%

100%

KF435981

1

43 css034

100%

100% Colletotrichum gloeosporioides EU732735

1

44 css035

100%

100% Epicoccum sorghinum

FJ427077

2 css036

45 css037

100%

100% Alternaria tenuissima

AY154712

1

46 css038

100%

AF071327

1

47 css039

100%

48 css041

99%

49 css043

100%

99% Cochliobolus intermedius 100% Cercospora ariminensis

KF251297

1

99% Toxicocladosporium rubrigenumFJ790287

1

EF419969

1

100%

50 css044

100%

100% Lophodermium conigenum

FJ861975

1

51 css045

100%

100% Guignardia mangiferae

JN791606

1

52 css046

100%

100% Macrophomina phaseolina

KF234547

1

53 css047

100%

HQ608103

1

99% Ochroconis humicola

RFLP

42 e50as034, e52as032

e50as001, e50as003-004, e50as010, e50as013-014, e50as023, e50as029, e50as033, e50as036, e50as041-042, e52as001, e52as004, e52as006010, e52as013, e52as015-016, e52as020-021, e52as024-026, e52as028-029, e52as031,e52as033-

11

e50as007, e50as017, e50as022, e50as030, e50as038, e50as040, e52as012, e52as030, e52as039, e50as047-048

1 e50as048

1

FJ481160

AB670716

>99% ITS2

e50as005-006, e50as009, e50as011012, e50as015-016, e50as018-021, e50as024-028, e50as031-032, e50as035, e50as037, e52as002003, e52as005, e52as011, e52as014, e52as017-019, e52as022e50as002, e50as031, 023, e52as027, e50as043-044, e50as046, e52as038, e52as045-046 40 e50as045

1

100% Nigrospora oryzae

98% Virgaria nigra

caw015-022,

1

AJ390436 JF502454

100%

100%

caw001-009, 22 caw011-014

2 caw056

100%

41 css032

caw029-030, caw032-035, caw038,

2 caw053

28 css006

99% Rosellinia subiculata

#

1

JQ760550

93%

RFLP

e50ss006-007, e50ss010, e50ss012, e50ss020, e50ss033, e50ss039-040, e50ss042, e50ss045046, e52ss001, e52ss003, e50ss003, e50ss022- e52ss005, e52ss007, e52ss009, e52ss018-019, e52ss025-027, 023, e50ss026, e50ss031, e52ss023, e52ss040-041, e52ss044, e52ss047048 33 e52ss028

3 caw047, caw048

1 csw034

FJ917287

27 css003

100%

>99% ITS2

1

2 css013, css014

100%

100%

cas026-037

AM749939

100%

33 css024

37 cas001-025

cas081, cas082, 4 cas084, cas085,

25 caw091

99% Hypoxylon perforatum

#

1

26 caw093

99%

1 caw089

2 caw041

HM122974 HQ608063

caw023-028, caw031, caw03617 037, caw039,

1

97% Nodulisporium hinnuleum

14 caw040

RFLP

3 caw044-046

1

99% Fusarium incarnatum

13 caw010

>99% ITS2

TE D

12 cas097

1 css042

EP

92% 100%

#

cas059, cas061065, cas067

AC C

10 cas091 11 cas096

HF674740

RFLP

Clone Adult

1

4 cas074

97%

Clone Seedling

Culture Adult Winter

RI PT

2 cas039

98%

Culture Adult Summer

SC

1 cas038

Culture Seedling Summer Culture Seedling Winter

M AN U

ITS2 or ITS1 Representativ coverag identity e >90% >95% Species OTU e isolate

5 cas075-078 1 csw012

1 caw086 2 caw095, caw096

cas079

1 e50ss025

ACCEPTED MANUSCRIPT

54 css048

100%

55 css049

100%

93%

JX496083

1

56 css050

100%

1

1 caw043

98%

KF435461

1

1 caw094

57 csw002 58 csw004 59 csw005

100%

99% Peniophora incarnata

JF439504

100%

99%

HQ914856

1

100%

95% Rhizosphaera kalkhoffii

AF013232

1

60 csw006

100%

61 csw007

100%

100% Cercospora poae

97% Teratosphaeria pseudafricana

DQ303008

62 csw009

100%

100% Cladosporium cladosporioides GQ221852

63 csw013

100%

100% Mycosphaerella marksii

64 csw014

100%

65 csw015

100%

66 csw016

100%

67 csw017

100%

68 csw018

98%

98% Leptosphaeria microscopica

FN386274

1

69 csw019

100%

100% Mycosphaerella mozambica

EU514257

1

70 csw020

100%

100%

JN695536

1

AF181701

1

GU237867

1

KF777180

1

JN198479

1

99% Phaeomoniella effusa

GQ154598

1

98% Lophodermium australe

U92308

3 csw022, csw023

97%

HF674740

1

100%

GQ153028

1

74 csw026

100%

99% Podospora appendiculata

AY999126

5 csw027-csw030,

75 csw031

100%

95%

AY561209

1

76 csw032

100%

100%

JQ760987

1

77 csw033

98%

99% Nemania macrocarpa

GU292823

1

78 csw035

95%

99% Biscogniauxia atropunctata

AJ390411

1

79 csw036

100%

100% Ophiocordyceps sinensis

JN198448

3 csw037, csw038

80 csw039

100%

99% Diaporthe terebinthifolii

KC343217

2 csw040

81 csw042

100%

97% Nemania diffusa

AB625442

82 csw043

100%

100% Nigrospora sphaerica

6 css007-012, 4 css015-018

95%

100% Anthostomella sepelibilis

AY908990

95%

97% Anthostomella sepelibilis

AY908989

85 csw052

100%

98% Coniochaeta africana

GQ154539

1

86 csw053

100%

96% Cytospora chrysosperma

JX076951

1

87 csw054

100%

100% Fusarium incarnatum

KJ371106

1

88 csw055

100%

100% Rosellinia corticium

AY908999

2 csw056

89 csw057

100%

100% Hypoxylon petriniae

JQ009309

1

92 e50ss002

91%

93 e50ss004

100%

93% Lophodermium nitens

DQ068340

99% Malassezia dermatis

93% 100%

96 e50ss009

90%

94%

97 e50ss011

99%

96% Stereum ostrea

AB509662

98 e50ss015

100%

99% Penicillium chrysogenum

JQ434684

100%

98% Peltigera neorufescens 100% Rosellinia corticium

100% Epicoccum nigrum

AY257916

JN578611

100 e50ss019

100%

99% Aureobasidium pullulans

100%

99% Cladosporium sphaerospermumDQ336706

102 e50ss027

100% 100%

99% Leptosporomyces raunkiaeri 100% Sphaerulina amelanchier

JX188093 GU187528 KF251634

104 e50ss030 105 e50ss036

100%

106 e50ss041

100%

99% Monilinia seaverii 100%

Z73793 KF800696

107 e52ss002

100%

97% Cryptococcus arrabidensis

NR073225

108 e52ss004

100%

98% Stereum sanguinolentum

AY089730

109 e52ss006

100%

99% Talaromyces radicus

HM469413

110 e52ss010

95%

2 e50ss018, e50ss048

1 caw085,

e50ss014, e50ss034e52ss033, e52ss037 5 035

2 caw087, caw088,

1 e50ss001

1 cas087

AY908999

101 e50ss021 103 e50ss029

1

AY390285

94 e50ss005

99 e50ss017

15 caw070-084

91%

95 e50ss008

2 caw057, caw058 caw062-063, 7 caw067,

EP

100%

KC354595

4 cas092-095

1 1 1 2 e50ss038 1 1 1 3

3 e52ss030, e50ss032 1 1 1 1 1 3 e50ss016, e52ss017 1 1 3

99% Capnobotryella renispora

AY220613

111 e52ss016

90%

95% Astrosphaeriella bakeriana

JN846716

100%

99%

GU721870

113 e52ss029

100%

99%

KF800620

1

114 e52ss036

100%

99% Aureobasidium pullulans

KF225026

1

115 e52ss039

84%

1 1 caw061

100% 100%

100% Penicillium citreonigrum 89%

118 e52ss046

100%

97% Montagnula aloes

1 1

90%

116 e52ss043

e52ss012, e52ss022

1

112 e52ss020

117 e52ss045

e50ss013, e50ss028

e50ss047, e52ss014, e52ss015, e52ss031, e52ss034035, e52ss042 9 e52ss021

AC C

91 e50as039

99% Nemania diffusa

e50ss043-044, e52ss011, e52ss013, e52ss024, e52ss038

2 e50ss024, e52ss032

2 csw044 csw045, csw0476 050

83 csw046

96%

1 cas083

2 csw041

KC519729

84 csw051

90 e50as008

8 e52ss008, e50ss037

RI PT

73 csw025

2 csw008 3 csw010, csw011

SC

100%

99% Peyronellaea prosopidis 100% Phoma bellidis

1 1 css040

M AN U

94%

72 csw024

99% Septoria passerinii 100% Peyronellaea australis

KC677886

1

TE D

71 csw021

100% Paraconiothyrium brasiliense

1 DQ093691

1 1

NR_111757 50

57

97

95

1 96

96

ACCEPTED MANUSCRIPT

Identified

Found butNot not found named in GenBank 83

27

8

AC C

EP

TE D

M AN U

SC

RI PT

Supplemental Table 4. List of 118 99% ITS2 OTUs used to represent the 491 isolates and clones used in this study. Their most similar known or unknown GenBank species accessions are listed with their percent coverage and similarity. Eighty-three genotypes had greater than 95% similarity to identified species. Twenty-seven genotypes had highly similar sequences deposited in GenBank but were not i

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

es deposited in GenBank but were not identified to species. Eight genotypes had no similarities in GenBank.

ACCEPTED MANUSCRIPT

Supplemental Table 1. Operational taxonomic units (OTUs) based on ITS2 are indicated for each sampled season and sampling method for needles of seedling and adult P. taeda.

Culture Clone Culture

Adult Clone

99%

97%

95%

90%

Summer

35

33

29

27

Winter

37

34

32

29

Summer

34

29

28

Summer

18

17

17

Winter

25

Summer

6

27 15

24

23

20

6

6

6

Supplemental Table 2. Reference sequences used in phylogenetic analyses. An asterisk indicates species used in the backbone constraint tree (Suppl. Fig. 1).

 

Acantholichen pannarioides Alternaria alternata* Alternaria limoniasperae Annulohypoxylon nitens Anteaglonium globosum Anthostomella eucalyptorum Antrodia albida Aspergillus fumigatus* Asterodon ferruginosa Athelopsis subinoconspicua Aureobasidium pullulans* Biscogniauxia mediterranea Botryosphaeria ribis* Caliciopsis orientalis* Candida albicans* Capnodium citri

EP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

GenBank accession EU825952 DQ678082 KC584284 AB376819 GQ221876 DQ890026 AY515349 AY660917 HE650987 JN649326 DQ470956 GQ377490 DQ678053 DQ470987 X70659 AY004337

TE D

Species

AC C

4   5   6   7  

Season

RI PT

Seedling

ITS2 OTUs

SC

Host age

Sampling method

M AN U

1   2   3  

Class

Agaricomycetes Dothideomycetes Dothideomycetes Sordariomycetes Dothideomycetes Sordariomycetes Agaricomycetes Eurotiomycetes Agaricomycetes Agaricomycetes Dothideomycetes Sordariomycetes Dothideomycetes Eurotiomycetes Saccharomycetes Dothideomycetes

1  

ACCEPTED MANUSCRIPT

 

KC408375

Sordariomycetes

GQ154609 GQ154611 GQ154602 GU553353 AF181535 EU002810 DQ470944 AF279380 DQ923537 GU048583 AY548815 AF279382

Leotiomycetes Leotiomycetes Sordariomycetes Sordariomycetes Tremellomycetes Tremellomycetes Leotiomycetes Dothideomycetes Sordariomycetes Sordariomycetes Arthoniomycetess Arthoniomycetess

AF261539 AF279385 AY016360 GU183122 DQ823100 AF356694 KC834023 AY097325 FN377748 JQ256433 AF543786 JQ743604 AY292423 AB079590 AF284122 AY281095 JN673047 AB376728 AY436415

Agaricomycetes Lecanoromycetes Dothideomycetes Dothideomycetes Eurotiomycetes Leotiomycetes Leotiomycetes Sordariomycetes Dothideomycetes Geoglossomycetes Sordariomycetes Dothideomycetes Pucciniomycetes Dothideomycetes Leotiomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes

SC

RI PT

Eurotiomycetes Dothideomycetes Coniocybomycetes Dothideomycetes

M AN U

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

DQ823099 KF251804 JX000086 JN938884

TE D

22 23 24 25 26 27 28 29 30 31 32 33

EP

21

Capronia pilosella* Cercospora zebrina Chaenotheca brachypoda* Cladosporium sphaerospermum Colletotrichum gloeosporioides Collophora africana Collophora paarla Coniochaeta prunicola Coniolariella hispanica Cryptococcus arabidensis Cryptococcus sp. Cudoniella clavus* Curvularia brachyspora Cytospora diatrypelloidea Daldinia eschscholzii Dendrographa decolorans* Dendrographa leucophaea f. minor Dentocorticium sulphurellum Diabaeis baeomyces* Dothidea ribesia Epicoccum nigrum Exophiala dermatitidis* Fabrella tsugae Flagellospora curvula Fusarium lichenicola Fusicladium cordae Geoglossum barlae* Glomerella cingulata* Guignardia mangiferae Helicobasidium purpureum Hortaea werneckii Hymenoscyphus ericae Hypocrea schweinitzii Hypoxylon crocopeplum Hypoxylon haematostroma Lasiosphaeria sorbina

AC C

17 18 19 20

2  

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EP

SC

RI PT

Sordariomycetes Arthoniomycetess Sordariomycetes Lichinomycetes Leotiomycetes Agaricomycetes Dothideomycetes Leotiomycetes Leotiomycetes Exobasidiomycetes Exobasidiomycetes Pucciniomycetes Microbotryomycetes Leotiomycetes Pezizomycetes Sordariomycetes Sordariomycetes Dothideomycetes Dothideomycetes Dothideomycetes Agaricomycetes Agaricomycetes Cystobasidiomycetes Sordariomycetes Sordariomycetes Neolectomycetes Dothideomycetes Sordariomycetes Sordariomycetes Sordariomycetes Dothideomycetes Dothideomycetes Dothideomycetes Orbiliomycetes Orbiliomycetes Sordariomycetes Lecanoromycetes Lecanoromycetes

M AN U

AY436417 HQ454551 AF353604 AF356691 AY544644 GU187588 GU048602 KC283138 AY004334 AB070365 AY743607 L20283 AY213003 AY544670 AF279398 HM034865 HM034862 GQ852612 AF309584 NG_027579 AF291350 AF291353 AF131061 JQ862605 AB376826 AF279401 JQ746554 AF286411 GQ221861 GQ377482 KF282651 KF282662 KF282666 DQ470953 DQ470952 KF636760 AF286822 AY584657

TE D

Lasiosphaeris hirsuta Lecanographa dimleaenoides Lecythophora mutabilis Lempholemma polyanthes Leotia lubrica* Leptosporomyces raunkiageri Letendraea helminthicola Lophodermium conigenum Lophodermium pinastri* Malassezia dermatis Malassezia restricta Melampsora lini* Microbotryum reticulatum Monilinia laxa* Morchella esculenta* Muscodor albus Muscodor fengyangensis Mycosphaerella marksii* Mycosphaerella molleriana Myriangium duriaei Myxarium mesonucleatum Myxarium sp. Naohidea sebacea Nemania bipallata Nemania diffusa Neolecta vitellina* Neosetophoma samarorum Neurospora crassa* Nigrospora oryzae Nigrospora sphaerica Ochroconis anellii Ochroconis sp. 1* Ochroconis sp. 2 Orbilia auricolor Orbilia vinosa* Paramicrothyrium chinensis Peltigera canina Peltigera degenii*

AC C

53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

3  

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Lecanoromycetes Lichinomycetes Lichinomycetes Eurotiomycetes Eurotiomycetes Eurotiomycetes Agaricomycetes Agaricomycetes Agaricomycetes Eurotiomycetes

AJ276065 FJ372408 GQ154618 AF450255 AF311045 JX463653 NG_027586 GU238046 AY512872 DQ883698 AY999103 HQ599593 JF440977 AY640962 AF189933 AF189962 EU552154 AF005702 AJ406401 Z19136 JX000094 FJ212339 AY254182 KF251746 AY346301 AF189984 AM269874

Arthoniomycetess Eurotiomycetes Eurotiomycetes Agaricomycetes Agaricomycetes Agaricomycetes Atractiellomycetes Dothideomycetes Atractiellomycetes Lecanoromycetes Sordariomycetes Sordariomycetes Sordariomycetes Eurotiomycetes Microbotryomycetes Cystobasidiomycetes Eurotiomycetes Saccharomycetes Atractiellomycetes Schizosaccharomycetes Coniocybomycetess Leotiomycetes Pucciniomycetes Dothideomycetes Sordariomycetes Cystobasidiomycetes Agaricomycetes

M AN U

SC

RI PT

AY257882 AF356693 DQ832334 AF034451 AY640958 HM469398 AF506425 AF287880 JX141469 AF353609

TE D

EP

101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127

Peltigera monticola Peltula obscurans Peltula umbilicata* Penicillium chrysogenum Penicillium freii* Penicillium variabile Peniophora incarnata Peniophora nuda Perenniporia japonica Phaeoacremonium chlamydosporum Phaeococcomyces nigricans Phaeomoniella capensis Phaeomoniella effusa Phellinus gilvus Phellinus wahlbergii Phlebia acerina Phleogena faginea Phoma bellidis Platygloea vestita Pleopsidium gobiense* Podospora appendiculata Pseudophloeospora eucalypti Pseumassariella vexata Pyrenula pseudobufonia* Rhodotorula hordea Rhodotorula pallida Rhynchostoma proteae Saccharomyces cerevisiae* Saccoblastia farinacea Schizosaccharomyces pombe* Sclerophora coniophaea Sclerotium cepivorum Septobasidium sp. Septorioides pini-thunbergii Sordaria macrospora Sporobolomyces foliicola Stereum hirsutum

AC C

91 92 93 94 95 96 97 98 99 100

4  

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SC

RI PT

Agaricomycetes Sordariomycetes Lecanoromycetes Agaricomycetes Dothideomycetes Geoglossomycetes Sordariomycetes Pezizomycetes Lecanoromycetes Ustilaginomycetes Ustilaginomycetes Ustilaginomycetes Microbotryomycetes Dothideomycetes Sordariomycetes Agaricomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Xylonomycetes Microbotryomycetes

M AN U

JQ746548 DQ377938 AY584647 AJ406478 AY016369 AY544653 JF704187 Z48319 DQ883692 AF009881 AJ236139 JN367335 AF009882 GU296206 AB740963 HM046925 JQ862643 AB376688 AY544648 AY761087 JQ838240 AF189905

Isolate 1 2 3 4 5 6 7 8

 

EP

Supplemental Table 3. Reference sequences from Arnold et al. (2007) used in our phylogenetic analysis. GenBank Accession KP215487 KP215490 KP215493 KP215494 KP215495 KP215505 KP215511 KP215520

AC C

8   9   10   11   12   13   14  

Stereum rugosum Taeniolella alta Teloschistes exilis* Thelephora vialis Trematosphaeria heterospora Trichoglossum hirsutum* Tubakia dryina Urnula hiemalis Usnea antarctica* Ustilago cynodontis Ustilago nuda* Ustilago sparsa Ustilentyloma fluitans Venturia populina* Virgaria nigra Vuilleminia cystidiata Xylaria badia Xylaria coccophora Xylaria hypoxylon* Xylomelasma sordida Xylona heveae* Zymoxenogloea eriophori

TE D

128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149

1734 1757 1814 1815 1817 1855 1894 1951

Class Leotiomycetes Leotiomycetes Sordariomycetes Leotiomycetes Sordariomycetes Dothideomycetes Dothideomycetes Dothideomycetes

5  

 

M AN U

SC

Sordariomycetes Leotiomycetes Atractiellomycetes Eurotiomycetes Dothideomycetes Sordariomycetes Sordariomycetes Leotiomycetes Sordariomycetes Sordariomycetes Dothideomycetes Sordariomycetes Leotiomycetes Sordariomycetes Dothideomycetes Dothideomycetes Sordariomycetes Sordariomycetes Dothideomycetes Sordariomycetes Atractiellomycetes

TE D

KP215622 KP215527 KP215624 KP215534 KP215539 KP215540 KP215553 KP215558 KP215562 KP215563 KP215569 KP215571 KP215573 KP215578 KP215580 KP215587 KP215592 KP215600 KP215601 KP215603 KP215607 KP215449 KP215450 KP215452 KP215453 KP215454 KP215455 KP215460 KP215462 KP215464 KP215465 KP215467 KP215468 KP215469 KP215472 KP215476 KP215479

EP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1981A 1990 2027A 2072 2092 2100 2151 2185 2204 2207 2231 2235 2247 2281 2294 2324 2350 2425 2448 2450 2617 Clone c0245 c0246 c0248 c0249 c0250 c0251 c0256 c0258 c0260 c0261 c0263 c0264 c0265 c0268 c0272 c0280

AC C

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

RI PT

ACCEPTED MANUSCRIPT

Dothideomycetes Dothideomycetes Microbotryomycetes Dothideomycetes Dothideomycetes Dothideomycetes Agaricomycetes Leotiomycetes Dothideomycetes Dothideomycetes Exobasidiomycetes Ustilaginomycetes Dothideomycetes Leotiomycetes Dothideomycetes Dothideomycetes

6  

ACCEPTED MANUSCRIPT

17

c0285

KP215480

Arthoniomycetes

15   16   17  

RI PT

Ustilago nuda Melampsora lini Schizosaccharomyces pombe Neolecta vitellina Saccharomyces cerevisiae Candida albicans Morchella esculenta Orbilia vinosa Peltula umbilicata

SC

Xylona heveae Chaenotheca brachypoda Sclerophora coniophaea Geoglossum barlae

Trichoglossum hirsutum Peltigera degenii Usnea antarctica

M AN U

Teloschistes exilis

Diabaeis baeomyces

Pleopsidium gobiense Capronia pilosella

Exophiala dermatitidis Penicillium freii

Aspergillus fumigatus

Pyrenula pseudobufonia Caliciopsis orientalis

Neurospora crassa Xylaria hypoxylon

Glomerella cingulata Monilinia laxa

Cudoniella clavus Leotia lubrica

Lophodermium pinastri Aureobasidium pullulans Mycosphaerella marksii Botryosphaeria ribis Venturia populina Alternaria alternata Ochroconis sp. 1

EP

TE D

Dendrographa decolorans

Supplemental Figure 1. Backbone constraint tree used for one of the three phylogenetic analyses of the nrLSU (Fig. 2). Relationships among fungal classes are conservative and based on several previously published multi-locus phylogenetic studies (see Materials and Methods).

AC C

18   19   20   21   22   23   24   25   26   27   28   29   30   31  

Us#laginomycetes/ Pucciniomycetes/ Schizosaccharomycetes/ Neolectomycetes// Saccharomycetes// Pezizomycetes// Orbiliomycetes// Xylonomycetes/ Lichinomycetes/ Coniocybomycetes/ / Geoglossomycetes// Lecanoromycetes/ / / / Euro#omycetes/ / / Sordariomycetes/ / Leo#omycetes/ / Arthoniomycetes/ / / Dothideomycetes//

Supplemental Table 4. See Excel File. List of 118 99% ITS2 OTUs used to represent the 491 isolates and clones used in this study. Their most similar known or unknown GenBank species accessions are listed with their percent coverage and similarity. Eighty-three genotypes had greater than 95% similarity to identified species. Twenty-seven genotypes had highly similar sequences deposited in GenBank but were not identified to species. Eight genotypes had no similarities in GenBank.

 

7  

ACCEPTED MANUSCRIPT

 

SC

M AN U

cas001 cas002 cas003 cas004 cas005 cas006 cas007 cas012 cas015 cas018 cas025 cas038 cas039 cas040 cas041 cas042 cas043 cas056 cas073 cas074 cas075 cas076 cas077 cas080 cas083 cas086 cas088 cas089 cas090 cas091 cas096 cas097 caw002 caw003 caw004 caw008

GenBank Accession KM519195 KM519196 KM519197 KM519198 KM519199 KM519200 KM519201 KM519202 KM519203 KM519204 KM519205 KM519206 KM519207 KM519208 KM519209 KM519210 KM519211 KM519212 KM519213 KM519214 KM519215 KM519216 KM519217 KM519218 KM519219 KM519220 KM519221 KM519222 KM519223 KM519224 KM519225 KM519226 KM519227 KM519228 KM519229 KM519230

TE D

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Genotype in phylogeny caw010 caw010 caw010 caw010 caw010 caw010 caw010 caw010 caw010 caw010 caw010 cas038 cas039 cas039 cas039 cas039 cas039 cas039 cas073 cas074 css044 css044 css044 cas080 csw025 cas086 cas088 cas089 cas090 cas091 cas096 cas097 caw010 caw010 caw010 caw010

EP

Genotype

RI PT

Supplemental Table 5. Isolates for which bidirectional sequences were obtained. Genotype naming code is as follows: “c” or “e” for culture or clone; “a” or “s” for adult or seedling; “s” or “w” for summer or winter; and “50” or “52” indicate annealing temperatures for clones. Example: csw057 is a cultured isolate from a seedling in the winter, e50ss015 is a clone using annealing temperature of 50º C from seedling needles from the summer.

AC C

32   33   34   35   36   37  

8  

 

SC

M AN U

KM519231 KM519232 KM519233 KM519234 KM519235 KM519236 KM519237 KM519238 KM519239 KM519240 KM519241 KM519242 KM519243 KM519244 KM519245 KM519246 KM519247 KM519248 KM519249 KM519250 KM519251 KM519252 KM519253 KM519254 KM519255 KM519256 KM519257 KM519258 KM519259 KM519260 KM519261 KM519262 KM519263 KM519264 KM519265 KM519266 KM519267 KM519268 KM519269 KM519270 KM519271 KM519272 KM519273 KM519274

TE D

caw010 caw010 caw010 cas039 cas039 caw040 caw042 cas080 caw049 caw049 caw051 caw052 caw054 caw055 caw055 caw059 caw060 e52ss016 caw069 caw090 caw091 caw093 css027 css003 css003 css006 csw043 caw052 caw052 csw046 css019 css020 css022 css023 css024 css025 css026 css027 css028 css029 css030 css031 css032 css033

EP

caw010 caw010 caw011 caw025 caw028 caw040 caw042 caw044 caw048 caw049 caw051 caw052 caw054 caw055 caw056 caw059 caw060 caw061 caw069 caw090 caw091 caw093 caw095 css003 css005 css006 css008 css013 css014 css017 css019 css020 css022 css023 css024 css025 css026 css027 css028 css029 css030 css031 css032 css033

AC C

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

RI PT

ACCEPTED MANUSCRIPT

9  

 

SC

M AN U

KM519275 KM519276 KM519277 KM519278 KM519279 KM519280 KM519281 KM519282 KM519283 KM519284 KM519285 KM519286 KM519287 KM519288 KM519289 KM519290 KM519291 KM519292 KM519293 KM519294 KM519295 KM519296 KM519297 KM519298 KM519299 KM519300 KM519301 KM519302 KM519303 KM519304 KM519305 KM519306 KM519307 KM519308 KM519309 KM519310 KM519311 KM519312 KM519313 KM519314 KM519315 KM519316 KM519317 KM519318

TE D

css034 css035 css037 css038 css039 css041 cas080 css043 css044 css045 css046 css047 css048 css049 css050 caw010 csw002 cas039 csw004 csw005 csw006 csw007 csw007 csw009 csw009 csw009 css045 csw013 csw014 csw015 csw016 csw017 csw018 csw019 csw020 csw021 csw021 csw021 csw024 csw025 csw026 csw026 csw026 csw026

EP

css034 css035 css037 css038 css039 css041 css042 css043 css044 css045 css046 css047 css048 css049 css050 csw001 csw002 csw003 csw004 csw005 csw006 csw007 csw008 csw009 csw010 csw011 csw012 csw013 csw014 csw015 csw016 csw017 csw018 csw019 csw020 csw021 csw022 csw023 csw024 csw025 csw026 csw027 csw028 csw029

AC C

81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

RI PT

ACCEPTED MANUSCRIPT

10  

 

SC

M AN U

KM519319 KM519320 KM519321 KM519322 KM519323 KM519324 KM519325 KM519326 KM519327 KM519328 KM519329 KM519330 KM519331 KM519332 KM519333 KM519334 KM519335 KM519336 KM519337 KM519338 KM519339 KM519340 KM519341 KM519342 KM519343 KM519344 KM519345 KM519346 KM519347 KM519348 KM519349 KM519350 KM519351 KM519352 KM519353 KM519354 KM519355 KM519356 KM519357 KM519358 KM519359 KM519360 KM519361 KM519362

TE D

csw026 csw031 csw032 csw033 caw052 csw035 csw036 csw036 csw036 csw039 csw039 csw042 csw042 csw043 csw043 csw046 csw046 csw046 csw046 csw046 csw046 csw051 csw052 csw053 csw054 csw055 csw055 csw057 e50as008 e50as039 e50ss002 e50ss004 e50ss005 e50ss008 e50ss009 e50ss011 e50ss015 e50ss017 csw042 e50ss019 e50ss021 e50ss027 e50ss029 e50ss030

EP

csw030 csw031 csw032 csw033 csw034 csw035 csw036 csw037 csw038 csw039 csw040 csw041 csw042 csw043 csw044 csw045 csw046 csw047 csw048 csw049 csw050 csw051 csw052 csw053 csw054 csw055 csw056 csw057 e50as008 e50as039 e50ss002 e50ss004 e50ss005 e50ss008 e50ss009 e50ss011 e50ss015 e50ss017 e50ss018 e50ss019 e50ss021 e50ss027 e50ss029 e50ss030

AC C

125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168

RI PT

ACCEPTED MANUSCRIPT

11  

ACCEPTED MANUSCRIPT

KM519363 KM519364 KM519365 KM519366 KM519367 KM519368 KM519369 KM519370 KM519371 KM519372 KM519373 KM519374 KM519375 KM519376

M AN U

38   39  

99% OTU

95% OTU

150 150

Seedling

100 100 50 50

107 107 Isolates

100" 100 100

50 50

50" 50 50

50"

0"

192 192 214 214

00

100

150 107 107 Isolates

192 214 192 214

000"

192

50"

OTUs

0" 0"

96107

Clones

Culture

AC C

150' 150

0

192 214

Clones

0

0"

96 107

Clones

99% 95% Series6' 90%

99% 95% Series6' 90% Series4'

Series5'

107 Isolates

192 192 214

Clone

50' 50

00'

192 192 214

Series4'

100' 100

192 192 214 214

50

50" 50"

96107

107 107 Isolates

100

50

50 0

100 100

150

EP

OTUs

100

Clone

150" 150 150

Adult

150 00

90% OTU

150 150

TE D

OTUs

Culture

RI PT

e50ss036 e50ss041 e52ss002 e52ss004 e52ss006 e52ss010 e52ss016 e52ss020 e52ss029 e52ss036 e52ss039 e52ss043 e52ss045 e52ss046

SC

e50ss036 e50ss041 e52ss002 e52ss004 e52ss006 e52ss010 e52ss016 e52ss020 e52ss029 e52ss036 e52ss039 e52ss043 e52ss045 e52ss046

192 214

Series5'

50 50' OTUs

169 170 171 172 173 174 175 176 177 178 179 180 181 182

0 0'

96 Clones

40   41  

 

12  

ACCEPTED MANUSCRIPT

Supplemental Figure 2. Rarefaction curves compared across 99%, 95%, and 90% ITS2 sequence similarity for samples from culturing and cloning. Seedling samples are in blue, adult tree samples are in green. Curves include black line representing observed OTUs, shaded area around curve representing the 95% confidence intervals, and dotted line representing bootstrap species richness.

RI PT

42   43   44   45   46   47  

Pine'clone'

Pine'clone'

Pine'clone'

e50as008'

caw010'

Pine'clone'

caw049'

M AN U caw049'

Pine'clone'

Pine'clone'

Pine'clone'

TE D

cas039'

Pine'clone'

cas039'

Pine'clone'

caw049'

100'bps'

EP

Supplemental Figure 3. Restricted fragment length polymorphism of a sample of adult clones digested with AluI and MspI. Digests were run on 2.5% agarose gel. Unique clones were sequenced to identify genotypes. Identified genotypes are labeled at the bottom.

AC C

48   49   50   51   52   53  

Pine'clone'

cas039'

SC

500'bps'

 

13  

ACCEPTED MANUSCRIPT

Culture

6

(22.3%) (6.4%)

(9.6%) (54.3%)

(7.4%)

2

0

(4.9%)

(0%)

27

6

2

(3.9%)

AC C

(52.9%) (11.8%)

4

RI PT (15.4%)

12

(7.8%) (23.5%)

13

(20.5%) (33.3%)

Culture

11

4 (9.8%)

Seedlings

Adult Trees

EP

Seedlings

8

6

24

(26.8%) (58.5%)

WINTER CULTURES

SUMMER CULTURES

c

3

Clone

51

9

7

9

(23.1%) (7.7%)

TE D

21

21

(16.4%) (31.3%)

(7.5%)

Clone

b

11

5

SC

8

M AN U

22

(32.8%) (11.9%)

54   55   56   57   58   59  

ADULT TREES Culture Culture Summer Winter

SEEDLINGS Culture Culture Summer Winter

a

23

6

(41.4%) (10.7%)

Adult Trees

8 (14.3%)

7

12

(12.5%) (21.4%)

Supplemental Figure 4. Venn diagram of endophytic fungi (OTUs = 99% ITS2 similarity groups) recovered from needles of P. taeda seedlings and adult trees using culturing and cloning methods in summer and winter seasons. The number and percentage of overlapping, nonoverlapping non-singleton, and non-overlapping singleton OTUs are represented in the circles.

 

14  

ACCEPTED MANUSCRIPT

60   61   62   63  

Outer circles represent singletons, OTUs that are found only once in the comparison. Inner circles represent non-singletons in the comparison.

RI PT

a 400 300

SC

200

0.97

0.91 0.925 0.94 0.955

M AN U

0.85 0.865 0.88 0.895

0.79 0.805 0.82 0.835

0.73 0.745 0.76 0.775

0.67 0.685 0.7 0.715

0

0.61 0.625 0.64 0.655

100

Simpson’s diversity

130

120

110

100

90

80

70

60

50

EP 40

30

20

0

Fisher’s alpha

AC C

64   65   66   67   68   69   70   71   72   73   74  

10

800 700 600 500 400 300 200 100 0

TE D

b

Supplemental Figure 5. Distribution of diversity indices from partitions of 48 random cultured isolates repeated 1000 times. Dotted lines indicate average diversity index of the 1000 random samplings. Arrows indicate diversity indices from cloning samples. Blue arrow and bars indicate seedling samples. Green arrow and bars indicate adult samples. a) Frequency distributions of Simpson’s index values; 0.96 ± 0.01 for seedling cultures; 0.81 ± 0.03 for adult cultures. b) Frequency distributions of Fisher’s alpha values; 66.41 ± 20.45 for seedling cultures; 8.20 ± 2.16 for adult cultures. Observed diversity from adult cloning samples was significantly lower than expected under the distribution of the 48 random cultured isolate partitions (Simpson’s diversity: t = 183.1, df = 999, p < 0.001; Fisher’s alpha: t = 99.1, df = 999, p < 0.001). Observed diversity from seedling cloning samples was significantly lower than expected under the distribution of

 

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the 48 random cultured isolate partitions (Simpson’s diversity: t = 538.5, df = 999, p < 0.001; Fisher’s alpha: t = 73.7, df = 999, p < 0.001).

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