Fungal Endophytes Associated with Three South American Myrtae (Myrtaceae) Exhibit Preferences in the Colonization at Leaf Level

fungal biology 118 (2014) 277e286

journal homepage: www.elsevier.com/locate/funbio

Fungal endophytes associated with three South American Myrtae (Myrtaceae) exhibit preferences in the colonization at leaf level

Aline B. M. VAZa,b, Andre G. F. C. DA COSTAc, Lucelia V. V. RAADd, Aristoteles GOES-NETOa,b,* aLaboratorio de pesquisa em Microbiologia (LAPEM), Departamento de Ci^encias Biologicas, Universidade Estadual de Feira de Santana, Feira de Santana, Bahia, Brazil bCentro de Pesquisas Rene Rachou (CPqRR), Fundac¸ao~ Oswaldo Cruz (FIOCRUZ), Belo Horizonte, Minas Gerais, Brazil cDepartamento de Estatıstica, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil dInstituto de Economia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil article info abstract

Article history: Fungal endophytes associated with Myrtaceae from Brazil and Argentina were isolated at Received 27 June 2013 three levels of nesting: leaf, individual host trees, and site collection. The alternating logis- Received in revised form tic regression (ALR) was used to model the data because it offers a computationally conve- 19 November 2013 nient method for fitting regression structures involving large clusters. The objectives of this Accepted 20 November 2013 study were to determine: (i) whether the colonization pattern is influenced by environmen- Available online 3 December 2013 tal variables, (ii) if there is some leaf part they prefer to colonize; (iii) if there is some fungal Corresponding Editor: endophyte aggregation between hierarchical levels; (iv) what the distance effect is on the Andrew N. Miller fungal association. The environmental variables were statistically significant only for Xy- laria, i.e., when the elevation and water precipitation increase and the temperature de- Keywords: creases, the odds ratio of finding another fungal endophyte of that genus previously Alternating logistic regression found increases. Sordariomycetes, Xylariales, and Xylaria exhibited leaf fragment preference Clustered responses to petiole and tip. Fungal endophytes showed association within leaf. The horizontal trans- Fungal distribution mission mode and the dispersal limitation may explain this association at the leaf level. Endophytic fungi Moreover, our results suggest that when a fungal endophyte infects a leaf or host tree in- Statistical modelling dividual, the odds ratio of dispersal inside them is greater. ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction fungal endophytes can be divided in clavicipitaceous and nonclavicipitaceous; the ﬁrst group infects some grasses Fungal endophytes inhabit healthy plant tissues during at and the second one can be recovered from asymptomatic tis- least one stage of their life cycle without causing any appar- sues of nonvascular plants, ferns and allies, conifers, and ent symptoms of disease or negative effects on the hosts angiosperms. The nonclavicipitaceous endophytes can be (Petrini et al. 1992). According to Rodriguez et al. (2009) the differentiated into three functional classes based on host

* Corresponding author. Laboratorio de pesquisa em Microbiologia (LAPEM), Departamento de Ciencias^ Biologicas, Universidade Estadual de Feira de Santana, Feira de Santana, Bahia 44036-900, Brazil. Tel.: þ55 (75) 3161 8296; fax: þ55 (75) 3161 8132. E-mail addresses: [email protected], [email protected], [email protected] (A. Goes-Neto). 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.11.010 278 A. B. M. Vaz et al.

colonization patterns, mechanisms of transmission between and requires the use of sophisticated approaches to statisti- host generation, in planta biodiversity levels, and ecological cal modelling. functions (Rodriguez et al. 2009). Endophytes occurring pri- Marginal models for temporally or spatially autocorre- marily in aboveground tissues (Class 3 endophytes, lated data sets model the marginal expectation of each bi- Rodriguez et al. 2009) are horizontally transmitted by spores nary variable separately as well as the association between and hyphal fragments from plant to plant, by biotic or abi- pairs of responses (Carey et al. 1993). To determine the rela- otic dispersion agents (Bernstein & Carroll 1977; Bertoni & tionship among responses with the explanatory variables, Cabral 1988; Saikkonen et al. 1998; Rodriguez et al. 2009). Liang & Zeger (1986) proposed the first generalized estimat- Moreover, class 3 endophytes form highly localized infec- ing equations (GEE1) approach to model the marginal mean tions, have the potential to confer benefits or costs on hosts (mean structure). Unfortunately, the dependence structure that are not necessarily habitat-specific, and are especially was considered a nuisance (Liang & Zeger 1986; Prentice notable for their high diversity within host tissues, plants, 1988). However, when the goal is estimating association pa- and populations (Saikkonen et al. 1998; Rodriguez et al. rameters, the second-order generalized estimating equations 2009). This group is composed predominantly by ascomyco- (GEE2) model gives more efficient results (Liang et al. 1992). tan fungi, and Sordariomycetes, Dothideomycetes, Pezizomycetes, However, GEE2 methods become computationally impracti- Leotiomycetes,andEurotiomycetes are the most frequently iso- cal if the number of measures within the cluster is large, lated classes (Rodriguez et al. 2009). Most of the fungal endo- e.g., higher than five (Carey et al. 1993). To solve these prob- phyte did not produce conidia or spores using the lems, the alternating logistic regression (ALR) procedure was conventional mycological media and the morphological proposed for simultaneously regressing the response on ex- identification is not possible. For this reason, the internal planatory variables and modelling the association among re- transcribed spacer (ITS1 and ITS2) and 5.8S regions of the sponses in terms of the pairwise odds ratio (Carey et al. 1993; nuclear ribosomal repeat unit have been used as the primary Ananth & Kantor 2004). There may be two objectives for fungal barcode marker to species delimitation (Schoch et al. modelling clustered data that include correlated responses 2012). using ALR (Dobson & Barnett 2002). The first one is to model The fungal endophyte composition reflects the interplay the mean structure as a function of covariates, which can be of host species, geographic distance, and climatic factors used for modelling the overall occurrence of fungal endo- (U’ren et al. 2012), with temperature and moisture being phytes. The second one is to model the dependence struc- the most important variables for explaining fungal diversity ture among pairs of binary responses, which can be used (Talley et al. 2002). Many host-associated microorganisms ex- for modelling the fungal endophyte association among clus- hibit patterns of genetic, morphological, and functional dif- tered data (Carey et al. 1993; Ananth & Kantor 2004). The ALR ferentiation that are related to the distribution of their is formulated based on the odds ratio, which is a particularly hosts (Papke & Ward 2004). Previous studies have shown dif- straightforward measure to capture the association between ferent patterns on host plant and/or tissue preference by binary or categorical outcomes (Liang et al. 1992; fungal endophytes varying from high (McKenzie et al. 2000; Molenberghs & Verbeke 2005). Su et al. 2010) to low tissue specificity (Cannon & Simmons In this paper, we studied the fungal endophytes at multiple 2002). hierarchical nesting levels using the ALR analysis. The study Multivariate binary responses are common place in eco- objectives included the following: (i) to assess, using the logical studies. Besides, the existence of multiple classes mean structure, whether the occurrence of fungal endophytes and levels of nesting in these ecological data is also com- at many distinct taxonomic levels is influenced by environ- mon. Repeated measurements within individuals tend to mental variables; (ii) to determine whether there is some be more similar than those taken among individuals, and leaf part preference in the fungal endophyte colonization; temporally or spatially proximate measurements are more (iii) to evaluate, using the dependence structure, whether fun- similar than temporally or spatially disparate ones (Fieberg gal endophytes at many distinct taxonomic levels exhibit as- et al. 2009). Traditionally, many of the commonly used statis- sociations within a leaf, individual host tree, and collection tics models in such studies (mainly regressions) assume in- site; and (iv) to appraise whether increasing the distance dependence among responses (Paradis & Claude 2002), among different individual host trees in the same collection which is not valid in temporally or spatially autocorrelated site will decrease the association of distinct taxonomic fungal data sets. One example of correlated responses is on longitu- groups. dinal studies where each subject is followed over a period of time in which repeated observations of the response and covariates variables are recorded (Carey et al. 1993). Since re- Material and methods peated observations are made on the same subject, observed responses are generally correlated (Pan & Connett 2002). In Study areas our work, the fungal endophytes were isolated following a hierarchical nesting: fungal endophytes occur in leaf frag- Three different sites were studied in Patagonia, Argentina, in ments of leaves, which are clustered within individual host the Andean Patagonian region. In Argentina, Luma apiculata trees that in turn occur in a site collection (Fig 1). In this (Myrtaceae) was collected at two different sites, Arrayanes for- case, as there is a hierarchical dependence among the vari- est (405104800S, 713605900W) and Puerto Blest (410103100S, ables, we can consider that they are correlated. This violates 714805800W), and Myrceugenia ovata var. nanophylla (Myrtaceae) the basic statistical assumption of independent observations was collected at one site, Espejo lake (404101800S, 714200000W). Preferences in the colonization at leaf level 279

Fig 1 e Fungal endophyte isolation from leaf samples of Luma apiculata, Myrceugenia ovata var. nanophylla, and Eugenia neomyrtifolia collected in Brazil and Argentina. The hierarchical nesting can be represented by the sets showed, where the lowest categories are included in the upper category levels successively: {Leaf} 3 {Individual host tree} 3 {Site collection}. The [ . [ . number of samples collected at each hierarchical level were: Site collection, Sc Sci1, ,Sci5; Individual host tree, Ih Ihi1, , [ . [ . Ihi20; Leaf, L Ii1, ,Ii5; Leaf fragment, Lf Lfi1, ,Lfi6.

These collection sites are situated in the Nahuel Huapi Na- Name assignment of molecular operational taxonomic units tional Park in the municipality of San Carlos de Bariloche, (MOTUs) Argentina. In Brazil, M. ovata var. nanophylla and Eugenia neomyrtifolia (Myrtaceae) were collected in two different sites of Pure cultures of the fungal isolates were grouped based on the an Atlantic rainforest area at the Centro de Pesquisas e Con- following morphological characteristics: (i) form, (ii) size, (iii) servaçao~ da Natureza Pro-Mata of PUCRS (292804400S, margin/border, (iv) surface, (v) colour, (vi) formation of aerial 501002500W), in Sao~ Francisco de Paula, Rio Grande do Sul mycelium, (vii) opacity, (viii) reverse colour. At least 50 % of state, Brazil. the fungal isolates of each morphospecies were identified by directly extracting their total genomic DNA and sequencing Fungal endophyte isolation the ITS region of the rRNA cistron. The extraction of DNA from filamentous fungi was performed according to Rosa Five apparently healthy leaves were collected from each of et al. (2009). The ITS domains of the rRNA cistron were ampli- 0 the 20 trees of all Myrtaceae species that occur in the studied fied using the universal primers ITS1 (5 -TCCGTAGGT- 0 0 sites. The trees were spaced approximately 5 m apart. All the GAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC- 0 leaves were stored in sterile plastic bags, and fungal isola- 3 ) as described by White et al. (1990). ITS amplification and se- tion was performed on the same day of the collection. The quencing were performed as described by Vaz et al. (2009). The leaves were surface-sterilized via successive dipping in ITS sequences obtained were analyzed in GenBank with 70 % ethanol (1 min) and 2 % sodium hypochlorite (3 min), BLASTn to search for similarity with the sequences deposited. followed by washing with sterile distilled water (2 min). Af- MOTUs were defined using a 97 % ITS region identity thresh- ter the leaf surface sterilization, six fragments (approxi- old (Edgar 2010; Sun et al. 2012). mately 4 mm2) were cut from each leaf: one from the base (C, near petiole), two from the middle vein (E and F), one The multivariate model from the left margin (D), one from the right margin (B), and one from the tip (A) (six leaf fragments/leaf; 30 leaf frag- The environmental variables, by principal component analy- ments/tree; 600 leaf fragments/site; 3000 leaf fragments sis (PCA), and the leaf fragments were considered in the overall) (Table 1, Fig 1). All the leaf fragments were plated mean structure in the ALR method. A PCA of the environmen- onto potato dextrose agar (PDA, Difco, USA) supplemented tal characteristics (elevation, water precipitation, and temper- with 100 mgml 1 chloramphenicol (Collado et al. 1996). The ature) at each site collection was performed (Table 2). Leaf plates were incubated at 15 C for up to 60 d. To test the ef- fragments were considered at the mean structure level to fectiveness of the surface sterilization, 100 ml of the water evaluate whether there is some leaf part preference for the used during the final rinse was plated on the PDA to test fungal endophyte colonization. The leaf fragment was not for epiphytic microbial contaminants. The binary responses considered at the dependence structure because it was the of the presence/absence of a fungal endophyte were consid- smallest sample unit. Thus it was not possible to make repli- ered for statistical analysis. cates, then, when considering the odds ratio of finding a fungal 280 A. B. M. Vaz et al.

fragment ¼ D) þ b I (leaf fragment ¼ E) þ b I (leaf Table 1 e Description of variables used for the 5 6 ¼ ALR statistical analysis. fragment F) (The C fragment was considered at the intercept.) Covariates Values Description (2) LogOR (Yj, Yk)¼ e a þ a Collection site 1 5 1 : Arrayanes forest 1I (collection site) 2 (distance jk) 2 : Puerto Blest (if j and k are different host trees in the same collec- 3 : Espejo lake tion site) 4 : ProMata a I (collection site) þ a I (individual host tree) 5 : ProMata 1 3 Host tree species 1e3 Luma apiculata (if j and k are different leaves on the same host tree) a þ a þ a Myrceugenia ovata 1I (collection site) 3I (individual host tree) 4I (leaf) var. nanophylla (if j and k are different leaf fragments on the same Eugenia neomyrtifolia leaf) Individual host treea 1e20 Leafb 1e5 The mean and dependence structures in our study were Fragmentc 1e6 C : Base (near petiole) modelled with the marginal odds ratio (Lipsitz et al. 1991). E : Middle vein upper F : Middle vein lower The association parameters were estimated by the odds ratio D : Left margin of finding another fungal endophyte of the same taxonomic B : Right margin level previously found at each one of the levels of nesting A : Tip (leaf, individual host tree, and site collection). A significantly Fungal endophyte Varies odds ratio suggests the presence of the aggregation of fungal numberd endophytes. The distance reflects the odds ratio of finding an- a Sampling was performed following a transect and each individ- other fungal endophyte of the same taxonomic level previ- ual host tree was spaced approximately 5 m apart. A total of 100 in- ously found when comparing two individual host trees dividuals from all host tree species was collected. spaced 1 m from each other. Despite the distance between b Five apparently healthy leaves were sampled randomly from each individual host tree species was approximately 5 m, each individual host tree resulting in a total of 500 leaves. the basic unit of distance for this statistical analysis is 1 m. c Six fragments were cut from each leaf and it was obtained 3000 leaf fragments overall. The ALR analysis was performed using the ordgee function d The fungal endophyte number isolates vary among the different from the geepack package (Yan 2002) and the PCA analysis us- species identified. ing prcomp function. All the analyses were carried out using the R program (R Development Core Team 2012) and the R script is available in Supplementary material. endophyte in one fragment, the comparison is performed with the odds ratio in another leaf fragment. In the dependence structure, the endophyte association at Results three distinct levels of nesting was considered (from the least inclusive until the most inclusive): leaf, individual host tree, A total of 939 fungal endophyte isolates were obtained from and site collection. Distance was also considered at the depen- 3000 leaf fragments and were identified in 51 distinct MOTUs. dence structure to represent how far each individual host tree Almost all taxa belonged to Ascomycota, with only Trametes be- was from each other in a same collection site. These variables longing to Basidiomycota. The most frequent fungal endo- were considered at the dependence structure because they phytes recovered belonged to Sordariomycetes and have dependence in their occurrence, i.e., one leaf was col- Dothideomycetes, accounting for 54.8 % and 38.9 %, respectively. lected from one specific individual host tree in a specific collec- Fungal endophytes belonging to the order Xylariales were the 3 3 tion site ({Leaf} {Individual host tree} {Site collection}, Fig 1). only taxon isolated from all host trees (Table 3). The first model (1) was used to estimate the influence of en- Figs 2 and 3 represent the probability of isolating one fun- vironmental variables and leaf part preference in fungal endo- gal endophyte belonging to their respective taxonomic group phyte occurrence at the mean structure whereas the second according to the environmental variables variation and leaf model (2) was used to estimate the association among the fragment. In Fig 2, the ordinate axis represents the mean per- levels of nesting. centage of isolating a fungus belonging to the distinct fungal taxonomic groups according to the first principal component ¼ ¼ b þ b þ b (1) Logit Pr (Y 1) 0 1I (PCA) 2I (leaf (PC1) from PCA analysis (abscissa axis). The environmental ¼ þ b ¼ þ b fragment A) 3I (leaf fragment B) 4I (leaf variables used in this study (temperature, elevation, and

Table 2 e PCA among environmental characteristics of each site collection. Components Elevation Water Temperature Standard Proportion Cumulative precipitation deviation of variance proportion (%)

PC1 0.709 0.623 0.330 1.338 0.597 59.7 PC2 0.007 0.475 0.880 1.005 0.336 93.3 PC3 0.705 0.621 0.341 0.448 0.066 100 Preferences in the colonization at leaf level 281

Table 3 e List of identiﬁed fungal endophytes from host tree species of Luma apiculata, Myrceugenia ovata var. nanophylla, and Eugenia neomyrtifolia from Andean Patagonian forest (Argentina) and Atlantic rainforest (Brazil). Identiﬁcation L1 L2 M1 M2 E1 Total

Sordariomycetes, Coniochaetales Coniochaeta velutina [JQ346221] 1 1

Sordariomycetes, Diaporthales Amphilogia sp. [JQ346197] 6 6 Diaporthe sp. 1 [JQ327869] 2 2 4 D. helianthi [JQ346194] 44 Diaporthe sp. 2 [JQ327871] 22 Diaporthe sp. 3 [JQ327870] 37 10 Diaporthe sp. 4 [JQ327872] 11 11 Diaporthe sp. 5 [JQ327871] 10 10 D. phaseolorum [JQ327873] 5 5 Diaporthe sp. 6 [JQ327874] 16 8 24 Diaporthe stewartii 66 Greeneria sp. 1 [JQ346195] 34 34

Sordariomycetes, Glomerellales Colletotrichum sp. 1 [JQ346206] 23 23 Colletotrichum boninense [JQ346207] 55 Colletotrichum sp. 2 [JQ346208] 77 Colletotrichum sp. 3 [JQ346209] 99 Colletotrichum sp. 4 [JQ346210] 20 20 Colletotrichum sp. 5 [JQ346211] 55 Colletotrichum sp. 6 [JQ346212] 22 22 Colletotrichum sp. 7 [JQ346213] 12 12 Colletotrichum sp. 8 33

Sordariomycetes, Hypocreales Cephalosporium sp. [JQ346222] 6 6

Sordariomycetes, Xylariales Annulohypoxylon sp. 3 [JQ327866] 2 2 Annulohypoxylon sp. 1 [JQ327864] 3 3 Annulohypoxylon sp. 2 [JQ327865] 13 13 Annulohypoxylon sp. 4 [JQ327867] 1 1 Biscogniauxia sp. 1 [JQ327868] 1 1 Nemania sp. [JQ327862] 3 27 30 Xylaria berteri [JQ327861] 33 17 50 Xylaria castorea [JQ327858] 2 5 7 Xylaria sp. 1 [JQ327859] 9 112 11 132 Xylaria enteroleuca [JQ327860] 46 46

Dothideomycetes, Botryosphaeriales Guinardia sp. 1 [JQ346219] 1 1 Guinardia mangiferae 37 21 58

Dothideomycetes, Dothideales Dothiora cannabinae [JQ346227] 4 4

Dothideomycetes, Capnodiales Cladosporium subtilissimum [JQ346203] 2 6 8 Cladosporium colombiae [JQ346204] 1 2 3 Mycosphaerella sp. [JQ346202] 192 192 Pseudocercospora basintrucata [JQ346205] 72 18 1 91

Dothideomycetes, Pleosporales Camarosporium brabeji [JQ346215] 1 1 Didymella sp. [JQ346225] 1 1 Lewia infectoria [JQ346214] 8 8 Microsphaeropsis olivacea [JQ346217] 1 1 Paraconiothyrium sp. [JQ346216] 1 2 3

Leotiomycetes, Helotiales Cryptosporiopsis actinidiae [JQ346199] 11 Mollisia cinerea [JQ346201] 1 1 Pezicula corylina [JQ346198] 34 34

(continued on next page) 282 A. B. M. Vaz et al.

Table 3 e (continued) Identiﬁcation L1 L2 M1 M2 E1 Total

Eurotiomycetes, Eurotiales Penicillium restrictum [JQ346224] 1 1

Pezizomycetes, Pezizales Peziza sp. [JQ346218] 1 1

Fungi incertae sedis, Mortierellales Mortierella sclerotiella [JQ346223] 1 1

Basidiomycota Trametes hirsuta [JQ346220] 15 15

L1: Luma apiculata (Arrayanes forest, Argentina); L2: Luma apiculata (Puerto Blest, Argentina); M1: Myrceugenia ovata (Espejo lake, Argentina); M2: Myrceugenia ovata (ProMata, Brazil); E1: Eugenia neomyrtifolia (ProMata, Brazil).

water precipitation) did not vary inside each site collection, var. nanophylla from Brazil, and Dothideomycetes more fre- and thus each point in the figure represents the PCA rotation quently associated with the temperate host tree species L. api- mean values obtained for each site collection: In Argentina; culata from Puerto Blest and Arrayanes forest. This same Luma apiculata in Arrayanes forest (0.63, first point), L. apicu- pattern maintained when considered the lowest taxonomic lata Puerto Blest (0.36, fourth point), Myrceugenia ovata var. level order and genus. The only exception happened with M. nanophylla Espejo lake (0.59, second point) and in Brazil: M. ovata var. nanophylla from Espejo lake (Argentina), which pre- ovata var. nanophylla ProMata (2.36, fifth point), Eugenia neomyr- sented similar occurrence of Xylariales, Xylaria, and Dothideo- tifolia ProMata (0.49, third point). The comparison at fungal mycetes fungi. class level showed that there was no overlapping in their oc- The ordinate axis in Fig 3 represents the mean percentage currence, with Sordariomycetes more frequently associated of isolating a fungus belonging to the distinct fungal taxo- with the tropical host tree species E. neomyrtifolia and M. ovata nomic groups according to the leaf fragment (abscissa axis). The leaf fragments A, C, and F exhibited the highest percentage of fungal endophytes belonged to the Sordariomycetes, Xylariales, and Xylaria. This pattern, however, was not observed for Dothideomycetes and Capnodiales.

Fig 2 e The graphs represent the probability of isolating one fungal endophyte belonging to its respective taxonomic group (Sordariomycetes, Dothideomycetes, Xylariales, Capno- diales or Xylaria) considering the first principal components (PC1) of the PCA of the environmental variables. Each point refers to an each site collection: In Argentina; Luma apiculata Fig 3 e The graphs represent the probability of isolating one in Arrayanes forest (L0.63, first point), Luma apiculata Pu- fungal endophyte belonged to its respective taxonomic erto Blest (0.36, fourth point), Myrceugenia ovata var. nano- group (Sordariomycetes, Dothideomycetes, Xylariales, Capno- phylla Espejo lake (L0.59, second point) and in Brazil: diales or Xylaria) considering the leaf fragment: near petiole Myrceugenia ovata var. nanophylla ProMata (2.36, fifth point), (C), middle vein (E and F), left margin (D), right margin (B), Eugenia neomyrtifolia ProMata (L0.49, third point). and tip (A). Preferences in the colonization at leaf level 283

Fig 4 e The barplots represent the probability (ordinate axis) of isolating two fungal endophytes belonging to their respective taxonomic group (Sordariomycetes, Dothideomycetes, Xylariales, Capnodiales or Xylaria) in the same individual host tree and leaf (abscissas axis).

Figs 4 and 5 represent the dependence structure and depict presence/absence of fungal endophyte obtained from the same site collection. In Fig 4, the ordinate axis represents the probability of finding or not (yes or no) another fungal endophyte of the same taxonomic level previously found when comparing the same individual host tree and leaf (abscissa axis). The probability was higher in the same leaf than in the same individual host tree. Fig 5 shows the probability of finding another fungal endophyte of the same taxonomic level previously found when increasing the distance among different individual host trees in the same site collection. When increasing the distance between two host trees at the same site collection, there is no significant decrease or increase in the probability (Pr [Yj ¼ 1; Yk ¼ 1]). The ALR models were analyzed at all fungal taxonomic levels, by species, genus, family, order, class and the models converged to Sordariomycetes, Xylariales, Xylaria, Dothideomy- cetes, and Capnodiales (Table 4), which means that the program generated results only for these fungal groups. Xylaria was the only genus isolated from all the host plants studied. Many models that consider species, genus, family, order, and class do not converge because the fungal endophytes belonging to Fig 5 e The graphs represent the probability (ordinate axis) those taxonomic levels were isolated at only one collection of isolating two fungal endophytes belonging to their re- site and/or at low frequency. spective taxonomic group (Sordariomycetes, Dothideomycetes, Only the first PCA was considered in the mean structure, Xylariales, Capnodiales or Xylaria) with increasing distance explaining nearly 60 % of the variance (Table 3), and it was between two individual host trees. 284 A. B. M. Vaz et al.

statistically signiﬁcant only for Xylaria (odds ratio 1.5, P ¼ 0.003; , Table 4). In other words, when the elevation and water precipitation increase and the temperature decreases, the odds ratio of ﬁnding another fungal endophyte of the genus Xylaria previ- ee

nanophylla ously found increases. The second variable considered in the mean structure was the leaf fragment. The odds ratios of the var. 1 3.46 1.38 8.67 leaf fragment were statistically signiﬁcant for Sordariomycetes, Xylariales, and Xylaria and ranged from 0.31 (0.21e0.44) to 0.71 (0.00e0.83). However, no differences in the distribution of Dothi- 0.001

-value O.R I.L. S.L. deomycetes and Capnodiales were found. At the dependence < 0.008 P structure, the odds ratios, in general, were statistically signiﬁ- cant at the leaf level for most of the groups, except for Xylariales,

Myrceugenia ovata and the individual host tree level was signiﬁcant only for Sordar- , iomycetes. The site collection and distance among host trees

ee were not statistically signiﬁcant for any group (Table 4). 1 3.07 1.52 6.22 Discussion Luma apiculata

Fifty-ﬁve distinct MOTUs were identiﬁed in this study based 0.001 -value O.R I.L. S.L. < 0.002

P on sequencing of the ITS region of rRNA, which is the accepted fungal DNA barcode region (Schoch et al. 2012). Many works have reported that fungal endophytes within Ascomycota en- compass the majority of foliar fungal endophyte species (Arnold 2007; Arnold & Lutzoni 2007; Johnston et al. 2012; ee Vaz et al. 2012; Langenfeld et al. 2013). Sordariomycetes and Xylariales are considered the dominant groups associated 0.66 0.53 0.83 0.637 1.25 0.50 3.12 0.161 1.76 0.80 3.88 0.46 0.30 0.72 0.452 1.29 0.66 2.53 0.121 1.62 0.88 2.98 0.36 0.24 0.54 0.364 1.43 0.66 3.13 0.073 1.95 0.94 4.03 0.46 0.32 0.66 0.839 1.09 0.48 2.47 0.141 1.54 0.87 2.75 0.61 0.48 0.79 0.592 1.25 0.56 2.79 0.193 1.65 0.78 3.50 1 1.50 1.15 1.95 0.942 1.02 0.64 1.62 0.798 1.08 0.60 1.95 2.66 1.41 5.03 with tropical hosts and the second most frequently fungal endophyte group recovered belonged to Dothideomycetes (Arnold & Lutzoni 2007; Higgins et al. 2007). This same pattern was ob-

0.001 served in our study since the fungal endophytes belonging to -value O.R I.L. S.L. 0.000 0.001 0.000 0.000 0.000 < 0.003 0.003 P those groups were the only ones isolated from all host tree species in all collection sites. The affinity between fungal endophyte and hosts varies according to the substrate chemistry, which influences the outcomes of interactions (biochemical inhibition, competition, avoidance, no response) ee (Arnold et al. 2003; Rajala et al. 2013). There is no overlapping in the curves representing the probability of isolating one fungal 0.71 0.00 0.83 0.50 0.00 0.73 0.37 0.00 0.53 0.45 0.00 0.64 0.64 0.23 0.80 1 endophyte belonging to its respective taxonomic group (Fig 2), suggesting some host specificity in the fungal endophyte colonization. This host specificity may be caused by the host leaf 0.001

-value O.R I.L. S.L. chemistry and the biological interactions. 0.000 0.000 0.000 0.000 0.000 < P The pattern of ﬁnding a higher probability of infection near the petiole than in more distal leaf fragments has previously been demonstrated (Bernstein & Carroll 1977; Wilson & Carroll 1994; Cannon & Simmons 2002). According to Wilson ee & Carroll (1994), the petiole end of the leaf expands less than the more distal two-thirds of the leaf. Consequently, infections that are established in the more distal leaf parts before 0.50 0.31 0.82 0.49 0.36 0.66 0.31 0.21 0.44 0.40 0.26 0.61 1 2.45 2.04 2.93 0.108 2.64 0.81 8.63 1.94 1.18 3.20 0.289 5.59 0.23 134.48 0.136 4.80 0.61 37.71 0.129 3.52 0.69 17.92 0.155 3.90 0.60 25.42 the leaf is fully expanded will become ‘diluted’ as leaf blade from the Andean Patagonian forest (Argentina) and the Atlantic rainforest (Brazil). Sordariomycetes Xylariales Xylariaexpands Dothideomycetes more compared Capnodiales with the petiole segments. The result is a leaf with more dense infections at the petiole end 0.006 0.000 0.000 0.000 0.001 0.245 1.81 0.67 4.93 0.848 1.40 0.04 44.98 0.875 1.25 0.07 21.22 0.589 2.01 0.16 25.42 0.564 2.73 0.09 83.30 0.000 0.009 -value O.R I.L. S.L. P (Wilson & Carroll 1994). Considering our results, we suggest

) that Sordariomycetes, Xylariales, and Xylaria most likely 3 ) 1 a

a exhibited some preference for leaf tissue colonization (Fig 3, ) 0.262 0.99 0.98 1.00 0.444 1.01 0.99 1.03 0.915 1.00 0.99 1.01 0.389 1.00 1.00 1.01 0.311 1.01 1.00 1.02 ) The ALR statistical analysis considering the fungal endophyte levels of genus, order, and class isolated from

2 Table 4). Probably, the highest odds radios found near the pet- b a e ) 0.091 1.34 0.95 1.87 0.363 0.88 0.66 1.16 iole and gradually decreasing towards the tip can be explained 1 ) 4 b a Eugenia neomyrtifolia F E D B A 0.082 0.60 0.34 1.07 by the leaf developmental pattern and reﬂect the ‘dilution’ of ¼ ¼ ¼ ¼ ¼ the fungal endophyte community. The somewhat greater col- Dependence structure Intercept (Site collection Fr Fr Fr PCA1 ( Fr Fr Individual host ( Table 4 and Mean structure Intercept ( O.R.: Odds ratio; I.L.: Inferior limit; S.L.: Superior limit; Fr: Leaf fragment. Signiﬁcant values in bold. Distance ( Leaf ( onization of the leaf tip region compared with the midrib and Preferences in the colonization at leaf level 285

margin fragments could possibly be explained by the leaf tip references being more prone to colonization, as rainwater draining off at the apex would tend to wash unestablished fungal propagules on the leaf surface towards the leaf tips (Wilson & Ananth CV, Kantor ML, 2004. Modeling multivariate binary re- Carroll 1994). However, this cannot be considered to explain sponses with multiple levels of nesting based on alternating the Dothideomycetes and Capnodiales distributions because logistic regressions: an application to caries aggregation. e there is no statistically significant leaf fragment colonization Journal of Dental Research 83: 766 781. Arnold EA, 2005. Diversity and ecology of fungal endophytes in pattern, suggesting that apparently there is no preference in tropical forests. In: Deshmukh S (ed.), Current Trends in Myco- the leaf fragment colonization for these groups. logical Research. Oxford & IBH Publishing Co. Pvt. Ltd., New The endophytes of woody plants are horizontally trans- Delhi, pp. 49e68. mitted by hyphal fragmentation and/or spores from plant Arnold EA, 2007. Understanding the diversity of foliar endophytic to plant (Faeth & Hammon 1997; Arnold et al. 2000; Arnold fungi: progress, challenges, and frontiers. Fungal Biology Review e 2005) and may be released passively by herbivores or physical 21:51 66. agents such as wind or rain (Rodriguez et al. 2009). Thus, the Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA, 2000. Are tropical endophytes hyperdiverse? Ecology Letters 3: 267e274. fungal endophyte colonization depends on the availability Arnold AE, Mejıa LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, and viability of fungal propagules in the surrounding envi- Herre EA, 2003. Fungal endophytes limit pathogen damage in ronment (Schulz & Boyle 2005). This mode of transmission a tropical tree. 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