Sapwood-inhabiting mycobiota and Nothofagus tree mortality in Patagonia: Diversity patterns according to tree species, plant compartment and health condition

Forest Ecology and Management 462 (2020) 117997

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Sapwood-inhabiting mycobiota and Nothofagus tree mortality in Patagonia: Diversity patterns according to tree species, plant compartment and health condition

T

Lucía Molinaa,b, , Mario Rajchenberga,b, Andrés de Errastia,b, Mary Catherine Aimec, María Belén Pildaina,b ⁎

a b c

Forest Protection Area, Centro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP), Ruta 259 Km 16.24, CC14 (9200), Esquel, Chubut, Argentina Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina Department of Botany & Plant Pathology, Purdue University, 915 W State St, West Lafayette, IN 47907, USA

ARTICLE INFO

ABSTRACT

Keywords: Fungal community Forest decline Water stress Latent pathogens Endophytes Temperate forests

Temperate forests ecosystems are threatened by declines and diseases worldwide. Causes have been investigated during the last decades allowing, in some cases, to relate such deterioration to climate change or pathogens but, in many cases, causes have not yet been elucidated. In recent decades, a phenomenon of grouped mortality, whose etiology remains unknown, has been observed in the two most distributed Nothofagus species of North Patagonian forests. This study aimed to assess sapwood-inhabiting fungal diversity of N. dombeyi and N. pumilio trees in healthy and affected stands in order to determine whether health condition shape these fungal communities, to characterize such patterns and, to seek for the likely pathogens associated with tree damage. Seven sites in Los Alerces National Park (Chubut, Argentina), were sampled seasonally for two years. Eighty-eight fungal taxa were recovered and identified. Spatial heterogeneity across plant compartments was found, involving community composition and structure, with stem harbouring greater diversity than root. It was found that the sapwood in both Nothofagus species was inhabited by different fungal species, in high richness and showing different patterns according to health condition and plant compartment. Health condition was a stronger driver of N. dombeyi sapwood-inhabiting fungal community than it was for N. pumilio; the latter evidenced plant compartment as a stronger driver of such community. Nothofagus pumilio presented higher frequency of decomposition agents in both affected and healthy trees than it was in N. dombeyi, indicating a greater wood deterioration of its stands. More decomposition agents and potential pathogens have been found in symptomatic stands of N. dombeyi than in healthy stands, although their frequency patterns do not allow the inference of conclusions about the primary fungal agent causing tree mortality. Our findings suggest a secondary role of living-wood-inhabiting mycobiota in tree damage processes as an expression of tree stress caused by climatic factors.

1. Introduction The Andean Patagonian forests are one of the last global reserves of temperate forests with little anthropic alteration and valuable diversity (Arroyo et al., 1996). Among the 20 tree genera present, 90% of the total area is occupied by species of Nothofagus Blume (Donoso, 1993). Species of this genus possess great socio-economic and ecological importance due to high wood quality, wide distribution, their ecological role as a pioneer species in succession ability to establish pure stands, response to forest management, and industrial uses that have led to

domestication and breeding programs for cultivation (McQueen, 1977; Veblen et al., 1995; Donoso Zegers, 2006). Nothofagus dombeyi (Mirb.) Oerst. is the dominant species in the temperate-humid forests of North Patagonia, whereas N. pumilio (Poepp. & Endl.) Krasser dominates highaltitude stands in north and central Patagonia (Donoso, 1993). Up to 50% of the distribution of both species are protected in natural reserves. Among the major threats to the sustainability of Nothofagus species in Patagonian forests are those produced by global climatic change (Kitzberger et al., 2000) and, associated or not, the outburst of pests and diseases. Understanding the emergent pathologies is critical for

⁎ Corresponding author at: Forest Protection Area, Centro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP), Ruta 259 Km 16.24, CC14 (9200), Esquel, Chubut, Argentina. E-mail address: [email protected] (L. Molina).

https://doi.org/10.1016/j.foreco.2020.117997 Received 31 October 2019; Received in revised form 12 February 2020; Accepted 13 February 2020 0378-1127/ © 2020 Elsevier B.V. All rights reserved.

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successful action to mitigate damage and prevent the many environmental, social and economic impacts caused by deterioration of the health of Nothofagus forests (FAO, 2010). Nothofagus forests are threatened by declines and diseases, like other temperate forests ecosystems, in a global context of sanitary deterioration. Causes have been investigated during the last decades. In some cases, deterioration of temperate forests has been attributed to climate change or pathogens but, in many cases, causes have not yet been elucidated (Haavik et al., 2015; Hennon et al., 2006; Meentemeyer et al., 2008; Rizzo et al., 2002). Interrelations between biotic and abiotic factors make forest decline a complex phenomenon to study (Manion, 1981). Both Nothofagus species present distinctive situations of regressive death, characterized by differentiated space mortality patterns: random and grouped mortalities. The etiology of random mortality has been attributed to water stress associated with the climate change (Villalba and Veblen, 1998). In Chile, it has been described a syndrome named Southern Beech Decline (SBD) that affects stands of N. dombeyi, N. pumilio and N. betuloides (Mirb.) Oerst. with symptoms that include partially foliage death and presence of ambrosia beetle galleries (Coleoptera, Scolytinae) associated with portions of dead plant tissue (Aguayo Silva et al., 2008; Kirkendall, 2011) and, in some cases, Phytophthora de Bary species (Fajardo et al., 2017). However, etiology of SBD has not yet been established. A few studies have proven the relationship between N. dombeyi decline (due to water stress) and climatic events (droughts) at a regional scale, but only in forest-steppe ecotone areas (Suarez et al., 2004; Suarez and Kitzberger, 2008). Grouped mortality occurs in higher precipitation areas and is particularly severe in Los Alerces National Park (LANP), Argentina (a UNESCO World Heritage Centre), where it has been observed since the late 1990s (LANP internal reports, unpublished). It has been initially associated with a disease that does not correspond to the characteristics of water stress although no research has been made, to date, assessing the causes. However, in several other temperate forest species, research has shown that susceptibility to pathogen infections can be increased by decreases in humidity and water availability (Coyle et al., 2015; Mattson and Haack, 1987). In Nothofagus grouped mortality, affected individuals displayed various symptoms, including redding of foliage, chlorosis, progressive branches drying, defoliation, redding and necrosis of vascular tissues, bleeding and beetle galleries. In advanced stages, affected trees showed signs of decomposing agents such as mycelial fans and basidiomes. Progression mortality is characterized by initial decay, followed by the death of individuals, and the formation of patches of mortality that grow radially as new individuals are affected through time (Fig. 1). This space–time pattern resembles those given by biological agents that spread by short distances or contact. Previous studies have reported the presence of potential pathogens such as Armillaria novae-zelandiae (G. Stev.) Boesew. and Huntiella decorticans Errasti, Z.W. de Beer & Jol. Roux associated with dying or dead N. dombeyi trees in North Patagonian forests (de Errasti et al., 2015; Pildain et al., 2009; Pildain and Rajchenberg, 2013), but no further conclusions could be drawn regarding their roles in such mortality. On the other hand, nowadays it is known that there is a complex and still barely-known diversity of plant-associated microorganisms that impacts host plant fitness, health and nutrition in both positive and negative ways through complex dynamics (Baldrian, 2016; Terhonen et al., 2019). In woody tissue, such a microbiota seems to be dominated by fungal organisms due to the advantages of filamentous growth in living-wood substrata (Baldrian, 2016). The term endophyte has been historically given to the diversity of such plant-associated inhabitants of plant tissues when no apparent effect was observed or no clear function in plant was evident (Carroll and Carroll, 1978; Wilson, 1995). Through time, it has been understood that plant-inhabiting fungi were ubiquitous, that their interactions with plant hosts were dynamic, and their functional role fell along a continuum from mutualism, commensalism and parasitism even through an individual's lifetime (Saikkonen et al., 1998; Stone et al., 2004). Thus, apparently neutral endophytes have

shown to serve as antagonists against host pathogens (Rungjindamai et al., 2008), to become saprobes when tree host deads (Oses et al., 2008) or to become pathogenic to hosts, causing tree damage under particular circumstances (Hyde and Soytong, 2008; Ragazzi et al., 2003; Rinaldi et al., 2008; Tao et al., 2008). The term ‘endophyte’ has been revised in recent years given the revealed complexity of interactions between plant-inhabiting fungi with their hosts and the understanding that such a ‘neutral’ interaction may switch to commensal or pathogenic lifestyles, depending on various factors (Terhonen et al., 2019). In any case, living tree mycobiota have been shown to be important components of forest ecosystems and significantly contribute to their diversity (Saikkonen, 2007; Stone et al., 2004). However, very little is known about the interaction and roles of living tree mycobiota in forest declines, wood decay and tree mortalities (Baldrian, 2016) and this is particularly true for Southern Hemisphere. In fact, living tree mycobiota is scarcely known in Southern Temperate Forests. However, an increasing number of studies have assessed forest declines from a plant-associated mycobiota approach, and have found that such fungal communities vary along with tree health condition, allowing, in some cases, the identification of number of species potentially causing the damage (Gennaro et al., 2003; Giordano et al., 2009; Gonthier et al., 2006; Lygis et al., 2005, 2004, Ragazzi et al., 2003, 2001). The aims of this study were to describe living-wood-inhabiting fungal (LWIF) diversity of North Patagonian Nothofagus forests, to characterize diversity patterns across plant compartment and health condition in N. dombeyi and N. pumilio grouped mortalities and, also, to assess likely pathogens associated with tree damage. 2. Materials and methods 2.1. Study area and sampling design The study was conducted in Los Alerces National Park (LANP), Argentina (42°58′27.075″S 71°38′37.725″W), from May 2016 to April 2018. Sampling sites were selected among records of Nothofagus grouped mortality stands previously spotted by LANP staff (internal LANP reports, unpublished). These sites were located in areas of high conservation priority, only reachable by trekking or sailing plus trekking. Therefore, sampling site selection was made by prioritizing sites with a greater proportion of path access and setting a maximum reaching effort of 4 hours. Seven sites were defined, 3 sites for N. pumilio and 4 sites for N. dombeyi (Fig. 1). Sites consisted of Nothofagus stands with one or several grouped mortality patches. In each site, 5 symptomatic trees and 5 asymptomatic trees were selected per sampling, sampled and georeferenced. Each studied stand was mainly evenaged and sampled trees were chosen among those with similar DAPs. Symptomatic trees were chosen from the margins of the mortality patch, where radial growth of the patch is taking place as new individuals are affected (Fig. 1b). Living trees in the first stage of decline were chosen, defined as transparent crown, dry branches, chlorosis and defoliation in more than 25% of the crown. Asymptomatic trees were healthy-looking, with a crown cover of 75–100% and selected from outside the patch at a minimum distance of 80 m of its margins. From each selected tree, wood samples (sapwood) from stem (at breast height) and roots were collected. This sampling was repeated seasonally during autumn and spring from 2016 to 2018. Each individual was sampled for a single time in the study. Sample collection was performed using a 5 mm diameter increment borer which was surface sterilized with 70% ethanol (v/v) regularly, and from which an approximately 5 cm long sapwood sample was recovered. Samples were stored individually in plastic bags and kept at 4℃ until processing 12 h later maximum. 2.2. Living-wood-inhabiting fungi isolation Core samples were cut into 5 mm pieces of living sapwood tissue in 2

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Fig. 1. Nothofagus tree mortality and tree symptoms. (a) Sampling site locations in Los Alerces National Park. Circles represent N. dombeyi sites, triangles are N. pumilio sites (1: Mt. Alto El Petiso coihue; 2: Lake Krugger; 3: Lake Menendez; 4: Alerce River; 5: Mt Alto El Petiso lenga; 6: Mt. El Dedal; 7: Mt. El Riscoso). (b) N. pumilio grouped mortality in Mt. El Dedal, the growth margins of the patch are pointed out. (c) N. dombeyi grouped mortality in Lake Menendez. (d) drying branches and defoliation in symptomatic N. pumilio tree. (e) drying branches and defoliation in symptomatic N. dombeyi tree. (f) redding of vascular tissue in N. dombeyi. (g) bleeding in N. pumilio.

pairs ITS5/ITS4 (White et al., 1990) and amplified setting PCR conditions as described in Pildain and Rajchenberg (2013). For Basidiomycota isolates, the partial 28S large sub-unit of nuclear rRNA (LSU) was also amplified using LR0R/LR5 primer pairs (Vilgalys and Hester, 1990) and setting PCR conditions as described in Rajchenberg et al. (2015). Reactions were performed in a total volume of 25 µL, with 1 µL of template DNA, 9 µL of pure sterile water, 1.25 µL of 10 µM forward and reverse primer and 12.5 µL of Promega GoTaq Green Master Mix (Promega, Madison, WI, USA). All PCR products were sent to Beckman Coulter, Inc. (Danvers, MA, USA) to proceed with Sanger sequencing. Sequences were manually edited using BIOEDIT 7.2.5 (Hall, 1999) to obtain consensus sequences. Taxonomic assignment was performed by querying obtained sequences to the GenBank database by using the nucleotide-nucleotide blast search option (MEGABLAST) in the NCBI website (https://blast.ncbi.nlm.nih.gov/) (Altschul et al., 1997). Blast consensus above 90–98% were considered as genus level matches, whereas identities above 98% were treated as species level matches. For those isolates that were assigned to the same genus through this methodology, an additional criterion was applied to assess whether they would be identified in a single taxon or not. Sequences with up to 2% of blast consensus were grouped within the same species. Additionally, identities were confirmed and completed by aligning consensus sequences with downloaded sequences of known species from the genera and performing phylogenetic analysis conducted in MEGA 10.0.5 (Kumar et al., 2018). Sequences generated in this study were submitted to GenBank database (Table 1).

a laminar flow cabinet and surface sterilized by submersion in 70% ethanol (v/v) and flaming (Hoff et al., 2004). Four core pieces were placed into Petri plates containing two different culture media and incubated at 20–24 °C up to 4 months. A 2% dextrose corn meal agar was used with a 1% Neomycin-Penicillin-Streptomycin solution (Sigma-Aldrich, St. Louis, MO, USA) in order to exclude bacterial organisms (Gazis and Chaverri, 2010). Basidiomycota selective medium was prepared according to Worrall and Harrington (1992): Benomyl-DichloranStreptomycin 1.5% malt extract agar with 40 mg Benomyl, 20 mg Dichloran and 100 mg Streptomycin sulphate per liter. Growing mycelia were transferred immediately to 2% malt extract agar in order to obtain pure cultures and to 2% liquid malt to proceed to DNA extraction. 2.3. Morphological and molecular identification Pure cultures were first grouped into morphotypes according to macroscopic and microscopic features. Morphotypes were then classified into 5 broad categories in order to assign accurate PCR protocols. DNA extractions from pure cultures were carried out using DNeasy Power Plant Pro Kit (QIAGEN, Hilden, Germany) following the manufacturer's recommendations. The full ITS region of rDNA (ITS1, ITS2 and the intervening 5.8S RNA gene) was amplified. Filamentous yeasts were amplified using primer pairs ITS1/ITS4 (White et al., 1990), setting thermocycling conditions according to López et al. (2016). ‘Zygomycota’ isolates were amplified using ITS1/ITS4 universal primer pairs (White et al., 1990) setting PCR conditions according to Walther et al. (2013). Deuteromycota isolates were amplified under PCR conditions described in White et al. (1990) using ITS1F/ITS4 primer pairs (Gardes and Bruns, 1993, White et al., 1990). Non-sporulation cultures without clamp connections were amplified using universal pair primers ITS1F/ ITS4 (Gardes and Bruns, 1993, White et al., 1990) and following Lindner and Banik (2009) PCR conditions. Non-sporulating cultures with clamp connections were amplified and sequenced using primer

2.4. Diversity analysis Diversity of Nothofagus LWIG was evaluated by calculating the alpha diversity estimates across all samples: Shannon-Wiener and Simpson’s indices, Pielou’s evenness index, Chao-1 and observed richness. The effects of tree health condition (symptomatic / asymptomatic), 3

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Table 1 Absolute frequencies of the 88 taxa found in N. dombeyi and N. pumilio samples, discriminated by health condition (St: symptomatic, At: asymptomatic) and plant compartment (F: stem, R: root). For each taxon, ecological strategies found in literature and Genbank accession numbers of their ITS and LSU sequences are listed. Asterisks refer to taxa reported as pathogenic in the literature.

Aleurodiscus patagonicus Ambrosiozyma sp. Anthostomella sp. Anthracobia muelleri Anthracobia sp. Arambarria destruens * Armillaria sparrei * Armillaria umbrinobrunnea * Arthrinium arundinis * Arthrinium sacchari * Arthrinium sp. * Ascocoryne cylichnium Ascocoryne sarcoides Ascocoryne sp. Atheliaceae Aurantiporus albidus

Aureobasidium sp. Beauveria sp. Cadophora sp. * Capronia sp. Ceratocystidaceae * Chaetomiaceae Cladosporium sp. * Coniochaeta sp1 * Coniochaeta sp2 * Coprinellus sp. Cordycipitaceae Cosmospora sp. Curreya sp. * Cytospora sp. * Fistulina antarctica

Ganoderma australe * Gyromitra sp. Helotiales * Hyaloscypha sp1 Hyaloscypha sp2 Hypholoma frowardii Lachnum sp Laetiporus portentosus * Leptodontidium sp. * Leptographium gestamen Metapochonia sp. Meyerozyma caribbica Meyerozyma guilliermondii Microcera sp. Nemania sp. Nigrospora sp. *

GenBank accession no.

Ecological strategy

MT076081 MT076082 MT076083 MT076084 MT076085 MT076086 MT076087 MT076088 MT076089 MT062835 MT076090 MT076091 MT076092 MT076093 MT076094 MT076095 MT076096 MT076097 MT076098 MT076099 MT076100 MT076101 MT076102 MT076103 MT076104 MT076105 MT076106 MT076107 MT076108 MT076109 MT076110 MT076111 MT076144 MT076112 MT076113 MT076114 MT076115 MT076116 MT076117 MT076118 MT076119 MT076120 MT076121 MT076164 MT076122 MT076123 MT076124 MT076127 MT076128 MT076129 MT076130 MT076131 MT076132 MT048519 MT076133 MT076134 MT076135 MT076136 MT076137 MT062834 MT076138 MT076139 MT076140 MT076141 MT076142 MT076143 MT076145 MT076146

St

N. dombeyi At F

R

St

N. pumilio At F

R

xylophile endophyte

– 1

– 0

– 1

– 0

0 0

2 1

2 0

0 1

xylophile pyrophile pyrophile wood_rot wood_rot wood_rot endophyte endophyte endophyte xylophile

1 1 – 2 3 – 1 0 1 1

0 0 – 0 0 – 0 1 0 0

0 0 – 0 2 – 1 1 1 1

1 1 – 2 1 – 0 0 0 0

– – 0 – – 0 – – – 1

– – 1 – – 1 – – – 0

– – 1 – – 0 – – – 1

– – 0 – – 1 – – – 0

xylophile xylophile saprophytic wood_rot

2 3 – 2

1 0 – 0

2 1 – 2

1 2 – 0

– – 1 3

– – 0 1

– – 0 0

– – 1 4

saprophytic entomopathogenic saprophytic saprophytic saprophytic cellulolytic saprophytic pathogenic pathogenic saprophytic

– 1 2 0 – – 0 – – 3

– 0 0 1 – – 1 – – 0

– 1 2 1 – – 1 – – 3

– 0 0 0 – – 0 – – 0

1 – – – 0 0 – 1 0 1

0 – – – 1 1 – 0 1 0

0 – – – 0 1 – 0 1 0

1 – – – 1 0 – 1 0 1

parasitic endophyte pathogenic

– 1 4

– 0 1

– 0 5

– 1 0

0 – –

1 – –

0 – –

1 – –

pathogenic

2

1

1

2

1

1

1

1

wood_rot

3

3

5

1

4

4

7

1

wood_rot saprophytic saprophytic xylophile xylophile wood_rot

– 1 – – – 6

– 0 – – – 0

– 1 – – – 5

– 0 – – – 1

0 – 0 0 1 0

1 – 1 1 0 1

0 – 1 1 0 0

1 – 0 0 1 1

xylophile wood_rot

0 5

1 1

0 4

1 2

– –

– –

– –

– –

endophyte wood_stain endophyte endophyte

2 2 – 2

0 0 – 0

1 2 – 1

1 0 – 1

– 1 2 1

– 0 0 1

– 0 2 1

– 1 0 1

endophyte entomopathogenic xylophile

– 1 0

– 0 1

– 1 1

– 0 0

2 – 3

0 – 1

0 – 3

2 – 1

saprophytic

0

1

0

1

–

–

–

–

(continued on next page) 4

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Table 1 (continued)

Obba valdiviana * Oidiodendron sp1 Oidiodendron sp2 Ophiostoma nothofagi * Ophiostoma novae-zelandiae * Ophiostoma valdivianum * Ophiostomataceae sp. * Paecilomyces / Isaria Paraphoma sp. * Pezicula sp. * Phacidium sp. * Phanerochaete velutina / sordida Hymenochaetaceae sp1 Hymenochaetaceae sp2 Hymenochateaceae sp3 Phialocephala sp1 Phialocephala sp2 Phialophora sp. * Phlebia sp. Pholiota baeosperma Pholiota brunnescens Pholiota multicingulata Pleosporaceae sp1 * Pleosporaceae sp2 * Pleosporaceae sp3 * Pleurostoma sp. * Postia dissecta Postia pelliculosa *

Pseudoinonotus crustosus Pseudovalsaria sp. Rasamsonia sp. Sarocladium sp. * Sistotrema brinkmannii Sporothrix cabralii * Stereum sp. Tolypocladium album Umbelopsis changbaiensis

Umbelopsis nana-dimorpha Umbelopsis ramanianna Umbelopsis vinacea Xylariales

GenBank accession no.

Ecological strategy

MT076147 MT076148 MT076149 MT076150 MT076151 MT076152 MT076126 MT076153 MT076154 MT076155 MT076156

MT076157 MT076158 MT076159 MT076160 MT076161 MT076162 MT076168 MT076163

MT076125 MT076166 MT076173 MT076187 MT076167 MT076174 MT076175 MT076188 MT076169 MT076177 MT076176 MT076172 MT076178 MT076165 MT076179 MT076180 MT076181 MT062836 MT076170 MT076190 MT076191 MT076182 MT076186 MT076192 MT076193 MT076189 MT076183 MT076184 MT076171 MT076185

St

N. dombeyi At F

R

St

N. pumilio At F

R

wood_rot

3

0

2

1

–

–

–

–

saprophytic saprophytic xylophile xylophile xylophile xylophile saprophytic endophyte endophyte pathogenic wood_rot wood_rot wood_rot wood_rot dark_septate dark_septate dark_septate wood_rot wood_rot

– 0 2 2 – – 1 – – 1 1 1 – 0 1 – 2 1 1

– 1 0 0 – – 0 – – 0 0 0 – 1 0 – 0 0 0

– 0 2 2 – – 1 – – 1 1 1 – 1 1 – 2 1 0

– 1 0 0 – – 0 – – 0 0 0 – 0 0 – 0 0 1

1 1 – – 2 1 – 1 1 – – – 1 1 – 0 – – 2

0 0 – – 0 0 – 0 0 – – – 1 0 – 1 – – 2

0 1 – – 1 1 – 1 0 – – – 2 1 – 1 – – 1

1 0 – – 1 0 – 0 1 – – – 0 0 – 0 – – 3

saprophytic wood_rot saprophytic saprophytic saprophytic pathogenic wood_rot

– 2 4 1 1 – –

– 0 0 0 0 – –

– 1 2 1 0 – –

– 1 2 0 1 – –

0 – – – – 1 2

1 – – – – 0 0

0 – – – – 1 2

1 – – – – 0 0

wood_rot

3

0

1

2

9

1

4

6

wood_rot

–

–

–

–

0

3

2

1

xylophile

–

–

–

–

3

1

3

1

saprophytic saprophytic mycorrhizal saprophytic wood_rot

2 – 1 – 1

0 – 0 – 1

2 – 1 – 2

0 – 0 – 0

– 2 3 0 –

– 1 0 1 –

– 2 1 1 –

– 1 2 0 –

endophyte saprophytic

1 1

0 0

1 1

0 0

– 1

– 2

– 0

– 3

saprophytic

4

1

2

3

–

–

–

–

saprophytic saprophytic

4 1

0 0

2 1

2 0

– 3

– 5

– 0

– 8

xylophile

–

–

–

–

0

1

1

0

Nothofagus species and plant compartment (stem / root) were evaluated by calculating and comparing alpha diversity estimates for each sample (Kruskal-Wallis rank sum test) and for each level of these variables (Pearson's Chi-square test). Beta diversity of fungal communities among different levels of these variables was evaluated as well by using dissimilarity indices: Jaccard, Kulczynski, Bray-Curtis and Horn calculated with both quantitative (relative abundance) and binary data (presenceabsence). Differences in fungal community composition and structure among different tree health condition, Nothofagus species and plant compartments were tested by performing perMANOVA and ANOSIM analysis, which assess both composition and abundances by using a distance matrix of dissimilarity indices among all samples. Tests were

performed with three different distance methods (dissimilarity indices): Bray, Jaccard and Euclidean. Significant tests of data dispersion were performed in order to verify homoscedasticity, in those cases where it was rejected, only ANOSIM inferences were considered. Additionally, a species accumulation curve was performed. Moreover, LWIF assemblages from N. dombeyi and N. pumilio were evaluated separately with regard to alpha diversity and effects of health condition and plant compartment. The species were analyzed separately due to the fact that they inhabit different environments, occupy different niches and have non-overlapping altitudinal distributions. Furthermore, with regard to the understanding of the phenomenon of grouped mortality by the study of fungal communities, the etiology of 5

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the disease is unknown and could even constitute different phenomena with different etiologies for each species. The partition was further supported by results of previously described alpha and beta diversity analyses. In order to better understand LWIF community patterns, diversity analysis was complemented by a functional approach. For all recovered and identified taxa, a bibliographical search was performed, looking for ecological strategies and functional backgrounds. Each taxon was assigned with an ecological strategy (Table 1). The diversity analyses previously described were performed by considering categories generated by the bibliographical search. Statistical analyses and graphics were performed in RStudio (version 1.1.456 – © 2009–2018 RStudio, Inc.) with the packages ‘vegan’ (Oksanen et al., 2018) and ‘ggplot2′ (Wickham, 2016). 3. Results

Fig. 3. Rarefaction curve. Cumulative number of OTUs as a function of cumulative number of samples.

3.1. Isolation and preliminary identification of LWIF

species (xylophile and wood decay associated), the endophytic yeast Meyerozyma caribbica, the wood staining Leptographium gestamen and two Curreya and Sarocladium species with unknown or wide range of known ecological strategies. Also, a high frequency of ‘Zygomycota’ isolates were recovered, representing 11% of total isolates and were identified as four species of the genus Umbelopsis: U. vinacea, U. changbaiensis, U. ramanianna, and a species from the U. nana/dimorpha species complex (Fig. 2). Although they were among common species due to their high frequencies, this group was significantly more abundant in root samples (p = 0.004, Pearson’s Chi-square test). They are known as soil inhabiting fungi but also as tree pathogens (in the case of U. vinacea). Regarding the diversity of LWIF of N. dombeyi and N. pumilio at Los Alerces National Park, the total species richness was 88 with an evenness (J’) index of 0.93 and a Shannon-Wiener (H’) index of 4.14. These values are unusually high and are due to the many number of rare species found, suggesting a high diversity of LWIF, as evidenced in the rarefaction curve (Fig. 3). The latter did not reach an asymptote, suggesting that richness could be underestimated. The number of missing species was estimated by the Chao-1 formula as 139, with a standard deviation of 22.

A total of 296 stem and roots sapwood samples were collected between autumn 2016 and spring 2018 for both N. dombeyi and N. pumilio species. Forty-six percent (1 3 6) of the samples showed fungal growth with no significant differences on fungal occurrence between tree species, tree health condition or plant compartment (Pearson’s Chi-square test). Two hundred and ten isolates were recovered and were distributed in 88 operational taxonomic units (OTUs) which were identified: 35 to species level, 40 to genus level, 11 to family level and 2 to order level (Table 1). The 210 isolates approximated a log-normal distribution, with many rare taxa and several common taxa (Fig. 2). Basidiomycota and Ascomycota frequencies were very similar, representing 43% and 46% of the total isolates, respectively (Fig. 2). The unexpected high abundance of Basidiomycota isolates was mainly due to the high frequency of a few wood-rot fungal species such as Fistulina antarctica, Postia pelliculosa, Hypholoma frowardii, Aurantiporus albidus, Laetiporus portentosus, Armillaria sparrei, Obba valdiviana and Pseudoinonotus crustosus, rather than taxonomic richness (which was 26 species). Although wood-rot Basidiomycota were the most common taxa found, they were significantly more abundant in symptomatic trees than in asymptomatic (p = 0.027, Pearson's Chi-square test). In fact, xylophilous taxa in a broad sense were more abundant in those samples (p = 0.009, Pearson’s Chi-square test). Regarding Ascomycota, high OTU richness was found (46) with a high number of rare species (singletons). Within the common Ascomycota taxa found there were the pathogenic genus Cytospora, two species of Ascocoryne and a Nemania

3.2. Diversity analysis Symptomatic trees presented a greater alpha diversity than

Fig. 2. Distribution frequency of isolates and taxa. Left: absolute frequency and reported strategy of more common taxa found (three or more isolates). Right: percentages of isolates (above) and OTUs (below) by Phyllum. 6

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Table 2 Alpha and beta diversity estimates calculated for the whole set of data and for the different Nothofagus species (Nd: N.dombeyi, Np: N. pumilio), parts of the plant (F: stem, R: root) and tree health status (St: symptomatic, At: asymptomatic). Alpha diversity indices: Pielou’s Eveness index (J’), Shanon-Wiener diversity index (H’), Simpson diversity index (λ) and Chao-1 with its standard deviation (SD). Quantitative and binary (b) beta diversity indices: Jaccard (Jc), Kulczynski (K), Bray-Curtis (B) and Horn (H). Alpha diversity total Nd Np F R St At Nd St At F R Np St At F R

spp richness 88 58 48 68 48 67 41 49 15 48 24 31 29 29 30

Beta diversity J' 0.93 0.95 0.92 0.95 0.94 0.94 0.95

H' 4.14 3.81 3.56 3.99 3.62 3.96 3.51 3.72 2.64 3.71 3.10 3.20 3.20 3.71 3.10

λ 0.98 0.97 0.96 0.97 0.96 0.98 0.96 0.97 0.92 0.97 0.95 0.95 0.95 0.97 0.94

Chao-1 139 99 100 96 95 99 148 71 101 78 42 58 115 78 160

SD 22 9 29 13 25 16 62 13 48 16 13 18 63 16 108

shared spp.

Jc

Jcb

K

Kb

B

Bb

H

Hb

Nd vs. Np

17

0.853

0.807

0.743

0.674

0.744

0.676

0.637

0.676

F vs. R

29

0.744

0.671

0.575

0.488

0.592

0.504

0.508

0.504

St vs. At

21

0.808

0.761

0.596

0.590

0.678

0.615

0.505

0.615

Nd

St vs. At

7

0.871

0.877

0.740

0.695

0.771

0.781

0.690

0.781

F vs. R

15

0.835

0.771

0.717

0.627

0.717

0.627

0.661

0.627

St vs. At

12

0.782

0.750

0.638

0.600

0.642

0.600

0.521

0.600

F vs. R

11

0.835

0.771

0.717

0.627

0.717

0.627

0.661

0.627

Np

Fig. 4. Non-metric multi-dimensional scaling (NMDS) ordinal plots showing community differences according to (a) health status, (b) plant compartment, and (c) Nothofagus species. Singletons were not plotted. St: symptomatic, At: asymptomatic, F: stem, R: root, Nd: N. dombeyi, Np: N. pumilio.

asymptomatic trees (p = 0.009, Kruskal-Wallis; p = 0.013, Pearson’s Chi-square test) with a total species richness for each treatment of 67 and 41, respectively (Table 2). However, missing species estimation by Chao-1 formula suggested a greater underestimation of species richness in asymptomatic trees than in symptomatic (148 ± 62 and 99 ± 16 respectively), resulting in the opposite pattern. This is due to the great number of singletons and doubletons found in asymptomatic trees which is used by Chao-1 formula to calculate missing species. Also, community composition and structure showed differences among symptomatic and asymptomatic trees when considering the whole dataset (p = 0.022, perMANOVA). These results were also confirmed by beta diversity dissimilarity indices that were high across symptomatic and asymptomatic wood inhabiting fungi assemblages and 21 species were shared between both treatments (Table 2, Fig. 4.a). The proportion of ecological strategies of LWIF found in symptomatic and asymptomatic trees showed differences as well (p = 0.009, perMANOVA). Symptomatic trees presented greater relative abundances of wood-rotting and xylophilous taxa than asymptomatic ones, as well as a higher abundance of taxa registered as pathogenic in the literature (p = 0.002, Pearson’s Chi-square test). Alpha diversity of LWIF in sapwood from stem and roots were not significantly different when the whole dataset was analyzed. Species richness was 68 and 48 taxa for stems and roots, respectively. Missing species estimation was similar in both cases: 96 ± 13 and 95 ± 25 (Table 2). However, community composition and structure did show significant differences between stem and roots assemblages (p = 0.005, perMANOVA) with 29 shared species and dissimilarity indices of beta diversity with values exceeding 0.5 (Table 2, Fig. 4b). Also, a

heterogeneity of ecological roles was found between stem and root fungi assemblages (p = 0.002, perMANOVA). A greater relative abundance of xylophilous and wood-rotting fungi was found in stem sapwood than in roots while more saprophytic fungi were found in roots than in stems. No differences were found in abundances of reported pathogenic taxa between stem and root assemblages. All the estimates suggest that alpha diversity of LWIF is greater in N. dombeyi than in N. pumilio samples (p = 0.018, Kruskal-Wallis test). Species richness was 57 in N. dombeyi and 48 in N. pumilio with 99 ( ± 9) and 100 ( ± 29) missing species estimated, respectively (Table 2). Composition and structure were significantly different as well (p = 0.001, perMANOVA) (Fig. 4c). Among the 88 OTUs identified in the study area, only 17 species (20%) were found to inhabit both Nothofagus species, mostly wood-rotting fungi but also the pathogenic genus Cytospora or the root decay associated Sistotrema brinkmannii. On the other hand, 39 species (45%) were only found in N. dombeyi, such as the endophytic species of Arthrinium and the pathogenic Arambarria destruens. Likewise, 31 species (36%) were only isolated from N. pumilio, such as one species of the parasitic family Cordycipitaceae and two species of the tree pathogen genus Coniochaeta. Also, the proportions of fungal ecological strategies showed to be different between both Nothofagus species (p = 0.005, perMANOVA) and a greater relative abundance of reported pathogenic taxa was found in N. dombeyi than in N. pumilio (p = 0.001, Pearson’s Chi-square test). Beta diversity indices between N. dombeyi and N. pumilio assemblages were high and the highest among the variables considered (Table 2). Further statistical analyses were performed with partitioned N. dombeyi and N. pumilio dataset, in order to evaluate more accurately the other variables under 7

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Fig. 5. Venn diagram showing shared and exclusive species isolated from N. dombeyi samples according to tree health condition and plant compartment. Species in blue were isolated only from stem, species in red were found only in roots, shared species are shown in black. Asterisks refer to taxa reported as pathogenic in the literature. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

exclusively found in symptomatic trees we recovered the pathogenic species Ophiostoma valdivianum, the wood-stain Leptographium gestamen and the endophytic species Meyerozyma guilliermondii, one Pezicula sp., a Metapochonia sp. and one species from the tree pathogenic genus Coniochaeta. Among the asymptomatic trees, we recovered mostly saprobes, xylophiles and a few wood-rot fungi that were exclusively isolated only from them. In addition, fungal communities in symptomatic and asymptomatic trees were different in composition (p = 0.04, perMANOVA). Higher abundances of endophytes, wood-rot taxa and xylophiles were found in symptomatic than in asymptomatic trees (p = 0.049, Pearson’s Chi-square test), and higher abundances of taxa reported as pathogens were recovered from symptomatic trees as well. (See Fig. 6.) Regarding plant compartment in N. pumilio, species richness of LWIF was higher in stem (48) than in root (30) (p = 0.042, Pearson’s Chisquare test); however, Chao-1 index is suggesting that roots have been differentially undersampled than stem (Table 2). Composition and structure showed differences between plant compartment (p = 0.004, perMANOVA) and dissimilarity indices among stem and root assemblages were higher than for health condition (Table 2). Eleven species (23%) were found to be present in both stem and roots, 19 species (40%) were found to be isolated exclusively from root samples and 18 (38%) exclusively from stem (Fig. 6). Ecological strategy analysis indicated a heterogeneity of roles (p = 0.01, perMANOVA) with more saprobe abundance in roots than in stem and more wood-rotting and xylophilous fungi in stem than in roots.

investigation. Regarding N. dombeyi health condition, alpha diversity of LWIF was found to be greater in symptomatic than in asymptomatic trees (p < 0.001, Pearson’s Chi-square test) with species richness of 49 and 15, respectively (Table 2, Fig. 5). Community composition and structure showed differences for tree health condition as well (p = 0.024, perMANOVA). Beta diversity dissimilarity indices were high (Table 2). Only 7 species (12%) were found in both symptomatic and asymptomatic trees among which we have common wood-rot fungi such as Fistulina antarctica, Laetiporus portentosus and one Stereum species, the xylophilous Ascocoryne sarcoides, one saprophytic species from the Umbelopsis nana/dimorpha species complex, but also two species of Curreya and Cytospora genera, reported as pathogenic in the literature (Fig. 5). Many species were only found in symptomatic trees (42 species, 86% of total). Among them we found several pathogenic species such as Ophiostoma nothofagi, Ophiostoma novae-zelandiae, Arthrinium arundinis, one Phacidium sp., Arambaria destruens and the root-decay associated Sistotrema brinkmannii. Ecological strategies of taxa found in N. dombeyi showed heterogeneity (p = 0.013, perMANOVA) with greater relative abundances of endophytes, wood-rotting fungi and xylophiles in symptomatic than in asymptomatic trees. Also, more taxa reported as pathogenic were recovered from symptomatic trees (p < 0.001, Pearson’s Chi-square test). Comparison within root and stem in N. dombeyi showed a greater alpha diversity in the stem sapwood (p = 0.005, Pearson’s Chi-square test), with species richness of 48 and 24 taxa found, respectively (Table 2). Composition was significantly different as well (p = 0.048, perMANOVA) and dissimilarity indices were high but lower than they were for health conditions. No patterns in ecological role were detected between stem and root assemblages and relative abundances of taxa registered as pathogenic did not vary either. Fifteen species (26%) were found both in stem and roots: wood-rotting fungi, saprobes and endophytes, but also one species of the pathogenic genus Cytospora (Fig. 5). Alpha diversity estimates did not differ between levels of tree health condition in N. pumilio. LWIF assemblages from symptomatic and asymptomatic trees had similar fungal species richness (31 and 29, respectively) although both showed a great number of exclusive species (40% and 35%, respectively) (Table 2). Among the common species found there are several wood-rotting fungi, saprobes and the endophytic yeast Meyerozyma caribbica (Fig. 6). Regarding the species

4. Discussion Living-wood-inhabiting fungi (LWIF) is a field that still remains barely studied to date, receives much less attention than leaf-inhabiting fungi and this is particularly evident in temperate forests (Unterseher, 2011). Moreover, studies of living-tree diversity in Southern Temperate Forests are scarce. Thus, generating information in these matters is greatly positive because of the contribution to the better understanding and the improvement of discussions regarding results comparisons. 4.1. Diversity of living-wood-inhabiting fungi of Nothofagus forests The present study is a contribution to the knowledge of living-tree fungal diversity in Nothofagus forests which to date had only a few 8

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Fig. 6. Venn diagram showing shared and exclusive species isolated from N. pumilio samples according to tree health status and location in tree. Species in blue were isolated only from stem, species in red were found only in roots, shared species are shown in black. Asterisks refer to taxa reported as pathogenic in the literature. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

et al., 2013; Stone et al., 2004). Most fungal taxa recovered were ascomycetes although the number of isolates were equal between ascomycetes and basidiomycetes which were mainly wood-rotting taxa (Fig. 2). The predominance of Ascomycota is characteristic of LWIF according to many previous studies, even in those with specific basidiomycetes isolation methods (Gazis and Chaverri, 2010; Jin et al., 2013; Robles et al., 2015; Stone et al., 2004) and has been understood as a reflection of the low stress-tolerance of Basidiomycota in functional sapwood (Chapela and Boddy, 1988; Giordano et al., 2009). However, an unexpected high isolate percentage of basidiomycetes was also found in several studies of LWIF (Martin et al., 2015) and molecular detection approaches have suggested the underestimation of plant-inhabiting basidiomycetes in culture-dependent studies (Arnold et al., 2007; Rungjindamai et al., 2008; Zhang et al., 2010) and have proven the presence of wood-rot basidiomycetes as an endophytic latent phase in temperate forests trees (Parfitt et al., 2010). ‘Zygomycota’ isolates were also unexpectedly abundant compared to many other studies that reported very low or null frequencies of ‘Zygomycota’ taxa (Jin et al., 2013; Johnston et al., 2017; Robles et al., 2015). Nevertheless, greater abundance of ‘Zygomycota’ taxa was found in root samples rather than stem, which is a plant compartment usually under considered in living wood inhabiting fungi studies as mentioned. Wood-rotting and xylophilous fungi were the functional groups most frequently recovered from wood samples in both Nothofagus species. The presence of wood-rotting fungi in standing Nothofagus trees is well documented as hardwood decomposition agents (Cwielong and Rajchenberg, 1995; Rajchenberg, 2006), however, it is important to remark that samples in this study were taken from sapwood of living trees that lacked signs of any sort. Wood-rotting fungi were recovered differentially in symptomatic trees than in asymptomatic ones and, also, more taxa reported as pathogenic were found in symptomatic trees. Thus, their presence could be explained by the “latent infection hypothesis”, as proposed by Boddy and Rayner (1983), where symptomatic individuals are under water stress which decreases water

studies for Nothofagus leaves in New Zealand (Johnston et al., 2017) and for LWIF in Chilean Patagonia (Oses et al., 2008). The present study has found 88 taxa of LWIF in the two major Nothofagus species of Northern Patagonia, N. dombeyi and N. pumilio. Species richness was similar or higher than in other studies in temperate and tropical forests (Gazis and Chaverri, 2010; Giordano et al., 2009; Lygis et al., 2005; Ragazzi et al., 2001). Also, diversity indices were unexpectedly high compared to similar studies cited, but it might reflect or be partly due to the weight of singletons in its estimation. Since the comparison of species richness between different studies may be misleading given its high dependence on sampling design and isolation methodology (Gamboa et al., 2002; Sun et al., 2012), results of the present study may reflect the intense sampling and isolation efforts employed. Nevertheless, the species accumulation curve was not asymptotic, indicating that LWIF communities were not fully sampled. The estimated completeness percentage of taxa recovered was of 63%, similar to those obtained in other studies in temperate forests (Unterseher, 2011). On the other hand, fungal occurrence across all samples was low: less than half of the samples showed fungal growth in culture. Classical indicators used in this study suggest undersampling, but also, a remarkable LWIF diversity among both Nothofagus species and high percentage of richness completeness were observed along with a low rate of recovery of fungi from samples in culture. Thus, can the underestimation of richness in this system compensate for greater sampling? Or is a greater sampling effort the better way to improve richness/species detection? The undersampling of endophyte diversity is an unavoidable fact in cultivation-dependent approaches, which provides an irreplaceable but preliminary knowledge of species richness (Truong et al., 2017; Unterseher, 2011). Obtaining pure cultures is crucial in plant pathology approaches by allowing further pathogenicity studies but, also, bioprospection of metabolites, enzymes and other downstream applications from strain collections (Terhonen et al., 2019). Further ‘next generation’ cultivation-independent approaches will complement and improve the assessment of LWIF diversity in Nothofagus forests as demonstrated in other forestry studies (Johnston et al., 2017; Kemler 9

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content in their living tissues, allowing fungal infection by decomposition agents.

temperate forest tree species (Giordano et al., 2009; Lygis et al., 2005) and are consistent with the model mentioned above of weakened defense against infection and low moisture content in tissues of damaged trees to a level allowing fungal development (Boddy and Rayner, 1983). Nonetheless, any frequency pattern of fungal species found differentially in symptomatic trees suggests a primary pathogen hypothesis. The Patagonian species Armillaria sparrei was found associated with symptomatic N. dombeyi trees. The genus Armillaria includes species that are among the most common and important causes of root rot in woody plants, worldwide (Kile et al., 1991; Fox, 2000). However, although most Armillaria species have the potential to infect healthy and stressed trees, they differ in their pathogenicity according to the host and may also act as necrotrophs and saprobes (Hood et al., 1991; Kile et al., 1991). To date, native species in Patagonia were known to act as saprophytes in native forest and to be able to become aggressive pathogens of exotic tree species (Pildain et al., 2009). Among the four Armillaria phylogenetic lineages present in Patagonia, only two were found to become woody plant pathogens (Pildain et al., 2010), although specific pathogenicity studies for Patagonian species are lacking and much needed. For Armillaria sparrei lineage, pathogenicity has not been reported to date and this is the first report of its association with damaged standing Nothofagus trees. Further studies are needed to understand the role played by this fungus in N. dombeyi mortality. Also, the ophiostomatoid species Leptographium gestamen, Ophiostoma nothofagi and Ophiostoma novae-zelandiae were found in association with symptomatic trees. Ophiostomatoid fungi are considered important contributing factors of forest decline, and their role as tree pathogens of Northern temperate forests is well documented (Coyle et al., 2015). In Patagonia, they have been found affecting exotic tree species in Chile (Peredo and Alonso, 1988; Zhou et al., 2004). Leptographium gestamen has been found associated with declined Nothofagus trees (Aguayo Silva et al., 2008; de Errasti et al., 2016; Kirkendall, 2011) although pathogenicity tests were not performed for native tree species. Another species to highlight is Arambarria destruens, exclusively found in roots of symptomatic N. dombeyi trees. Arambarria destruens is a Patagonian wood-rotting pathogen that has been found involved in canker rot of native and exotic trees in the Southern Hemisphere (Pildain et al., 2017). Canker rots occur after functional sapwood is damaged by the heart-rot fungus (Vasaitis, 2013) although, in this study, frequency of Arambarria destruens in symptomatic trees does not allow the inference of such a primary pathogenic role in tree damage. The cubical heartrotting Postia pelliculosa was, also, exclusively associated with symptomatic N. dombeyi trees. This species is the most frequent and important wood-rotting fungus affecting N. pumilio standing trees and it has been hypothesized that it is able to infect Nothofagus trees through the roots as well as through the canopy (Cwielong and Rajchenberg, 1995). Besides, four Umbelopsis species were found associated with symptomatic N. dombeyi trees: Umbelopsis changbaiensis, Umbelopsis ramanianna, Umbelopsis vinacea and a species of the Umbelopsis nana/dimorpha complex. Species from the genus Umbelopsis are known as saprophytic soil inhabitant fungi but have also been found in sapwood of declined trees in temperate forests (Giordano et al., 2009; Hoff et al., 2004).

4.2. Diversity comparisons across plant compartment, Nothofagus species and health status LWIF assemblages, evidenced spatial heterogeneity across stem and root in both community structure and ecological strategies. The localized nature of endophyte infections and spatial community patterns across plant compartments have been thoroughly documented (Stone et al., 2004). Findings in the present study coincides with others regarding differential community structure between stem and roots (Jin et al., 2013; Qian et al., 2019; Sun et al., 2012) and a greater richness and diversity in stems than roots (Cregger et al., 2018; Durand et al., 2017; Terhonen et al., 2019), which has been understood to be caused by a more restrictive environment in roots compared with other tissues (Fisher et al., 1991) and cohabitation with ectomycorrhizal fungi (Kernaghan and Patriquin, 2011; Reininger et al., 2012; Sietiö et al., 2018). As expected, N. dombeyi and N. pumilio showed the greater dissimilarities in LWIF assemblages among the different variables considered (plant compartment and health condition), with very few shared species between hosts and different community composition and structure. Lower species richness was found in N. pumilio than in N. dombeyi, as well as lower diversity indices. Both species have distinctive nonoverlapping and nonadjacent altitudinal distributions in Northern Patagonia with N. pumilio dominating altitudinal treeline in pure stands (Daniels and Veblen, 2004). Elevational gradient has been described as a biogeographical factor whose variation encompasses species richness patterns for many living groups as an extension to Rapoport’s latitudinal rule to altitude, explaining lower species richness observed in higher elevations at landscape levels (Stevens, 1992). Several studies have reported elevation as an explanatory factor that shaped tree-inhabiting fungal communities in particular (Hashizume et al., 2008; Osono and Hirose, 2009; Siddique and Unterseher, 2016) and, furthermore, much has been written about host species as strong drivers in tree-inhabiting mycobiome at landscape and individual stand level (Arnold et al., 2007; Carroll, 1995; Johnston et al., 2017; Sun et al., 2012) sometimes explained as due to greater host preference in certain lineages (Ibrahim et al., 2016; Schlegel et al., 2018; Toju et al., 2019). Each Nothofagus species is inhabited by a different fungal community. Furthermore, these communities have been shown to vary according to host health condition and plant compartment by different relative magnitudes in each Nothofagus species. Whereas health condition more strongly shaped fungal communities in N. dombeyi, plant compartment accounted for greater dissimilarities in N. pumilio wood fungal communities. There are few previous studies that assess LWIF diversity from the forest pathology perspective that coincides in that fungal communities vary between damaged and apparently healthy trees. In some cases, communities shared composition but varied in structure and infection rates with higher frequencies in damaged trees (Gennaro et al., 2003; Ragazzi et al., 2003, 2001). In other cases, it has been reported that living wood in healthy and damaged trees was inhabited predominantly by different fungal species (Giordano et al., 2009; Lygis et al., 2005, 2004) which is the case in the present study.

4.4. Nothofagus pumilio. Diversity comparisons across health status and plant compartment

4.3. Nothofagus dombeyi. Diversity comparisons across health status and plant compartment

In the case of N. pumilio, LWIF communities between different health conditions and plant compartments had similar species richness but evidenced different species composition. As mentioned above, LWIF communities in this tree species showed the greater dissimilarities between plant compartments than between symptomatic and asymptomatic trees. Umbelopsis changbaiensis and Umbelopsis vinacea were exclusively present in roots, in high frequencies. Contrasting with N. dombeyi findings, Umbelopsis species could not be associated with any particular health condition in N. pumilio. Although Umbelopsis species, and ‘Zygomycota’ taxa in general, are not usually listed in wood

LWIF communities in symptomatic N. dombeyi trees had three times greater species richness than asymptomatic trees and greater diversity indices. Similar results have led to the hypothesis of species turnover in LWIF communities with changes in tree health condition (Lygis et al., 2004). In addition, among fungal taxa exclusively found in symptomatic trees (86% of total), there were many taxa reported as pathogenic in the literature. These results coincide with those from other studies in 10

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endophyte studies as mentioned above, they have been reported in several studies targeting roots of temperate forest trees and have been associated with both healthy and declined trees of temperate forest tree species (Amos and Barnett, 1966; Fisher et al., 1991; Giordano et al., 2009; Holdenrieder and Sieber, 1992; Holdenrieder et al., 1994). Umbelopsis species are common inhabitants of soils, common associates of wood and endophytes in root xylem tissue although their ecological roles in forest ecosystems remain undetermined (Hoff et al., 2004). Between health conditions, differences in species composition were not associated with the ecological roles of taxa present although some species exclusively found in symptomatic trees might be highlighted, such as Postia pelliculosa, Ophiostoma valdivianum and Leptographium gestamen. The scope of ophiostomatoid species in forest decline was already discussed above as well as P. pelliculosa background in N. pumilio decay. Although present in both treatments, P. pelliculosa was recovered in 90% from symptomatic trees. It was the most frequently encountered taxon. Despite the great dissimilarities between plant compartments in N. pumilio fungal communities, P. pelliculosa was isolated in high frequencies from both root and stem samples supporting the major importance of P. pelliculosa in N. pumilio wood decay (Cwielong and Rajchenberg, 1995). In contrasting to N. dombeyi, asymptomatic N. pumilio fungal communities included taxa that indicate a certain level of wood decay, such as the wood-rotting Ganoderma australe, Pseudoinonotus crustosus, Armillaria umbrinobrunnea, Hypholoma frowardii and the wood-staining ophiostomatoid Sporothrix cabralii. These differences in mycobiota patterns between Nothofagus species could be given by differential susceptibilities to decomposing agents or pathogens as has been demonstrated for many tree species of temperate forests (Desprez-Loustau et al., 2006). Moreover, such differences might respond, in a Boddy and Rayner, (1983) model, to a lower water content in living tissues of N. pumilio given site features and water availability according to altitudinal distribution (Daniels and Veblen, 2004) which have also been shown to drive greater susceptibilities to cavitation in Patagonian Nothofagus species (Bucci et al., 2012). In addition, wood-rotting fungi have been reported as a good tree age indicator in several species (Berry and Lombard, 1978; Lesica et al., 2003; Zagory and Libby, 1985) thus, higher frequencies of woodrotting fungi may indicate older stand ages in N. pumilio than in N. dombeyi. It is likely that N. dombeyi stands were recent secondary postfire successions, given the known history of human use for these lands until 150–100 years ago (Willis, 1914; Novella, 2018).

primary fungal agent causing tree mortality. Our findings suggest a secondary role of living-wood inhabiting mycobiota in tree damage processes as an expression of tree stress caused by climatic factors. CRediT authorship contribution statement Lucía Molina: Conceptualization, Methodology, Formal analysis, Data curation, Investigation, Writing - original draft, Visualization. Mario Rajchenberg: Conceptualization, Resources, Writing - review & editing, Supervision. Andrés Errasti: Methodology, Investigation. Mary Catherine Aime: Resources, Investigation. María Belén Pildain: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We thank Camilo H. Rotela, Martín Izquierdo, Cristina Agüero and Gabriel Bauer from the Conservation Department of Los Alerces National Park for their assistance in the investigation of non-formal background about grouped mortality in the national park. Also, we thank to the rangers Darío Barroso, Mauro Piñeiro, Marcelo Guisasola, Alejandro Mior and Gustavo Paramosz of Los Alerces National Park, for their invaluable assistance and experience in the field. Maximiliano Rugolo and Juan Monges kindly helped in the field, while Mariano Aquino contributed in Umbelopsis species identification and Jairus F. Chittenden helped with molecular tasks in the laboratory. We thank Geoffrey Williams for the language revision. Funding: this work was supported by the ANPCyT 2015/1933 (to MR), ANPCyT 2015/1723 (to MBP). References Aguayo Silva, J., Ojeda Alvarado, A., Baldini Urrutia, A., Cerda Martinez, L., Emanuelli Avilés, P., Kirkendall, L.R., Sartori Ruilova, A., 2008. Manual de plagas y enfermedades del bosque nativo en Chile Manual de Cooperación Técnica. FAO, Santiago de Chile. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Amos, J.K., Barnett, H.L., 1966. Umbelopsis versiformis, a new genus and species of the Imperfects. Mycologia 58, 805–808. Arnold, A.E., Henk, D.A., Eells, R.L., 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. https://doi.org/10.3852/ mycologia.99.2.185. Arroyo, M.T.K., Cavieres, L., Penaloza, A., Riveros, M., Faggi, A.M., 1996. Relaciones fitogeograficas y patrones regionales de riqueza de especies en la flora del bosque lluvioso templado de Sudamerica. In: Armesto, J., Villagran, C., Kalin, M. (Eds.), Ecologıa de los Bosques Nativos de Chile. Editorial Universitaria, Santiago de Chile, pp. 71–92. Baldrian, P., 2016. Forest microbiome : diversity, complexity and dynamics. FEMS Microbiol. 1–22. https://doi.org/10.1093/femsre/fuw040. Berry, F.H., Lombard, F.F., 1978. Basidiomycetes Associated with Decay of Living Oak Trees. For. Serv. Res. Pap. NE-413 46, 1–11. Boddy, L., Rayner, A.D.M., 1983. Origins of decay in living deciduous trees: the role of moisture content and a re-appraisal of the expanded concept of tree decay. New Phytol. 94, 623–641. Bucci, S.J., Scholz, F.G., Campanello, P.I., Montti, L., Jimenez-Castillo, M., Rockwell, F.A., Manna, L.La., Guerra, P., Bernal, P.L., Troncoso, O., Enricci, J., Holbrook, M.N., Goldstein, G., 2012. Hydraulic differences along the water transport system of South American Nothofagus species: do leaves protect the stem functionality? Tree Physiol. 32, 880–893. https://doi.org/10.1093/treephys/tps054. Carroll, G., 1995. Forest endophytes : pattern and process. Can. J. Bot. 73, S1316–S1324. Carroll, G.C., Carroll, F.E., 1978. Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Can. J. Bot. 56, 3034–3043. Chapela, I.H., Boddy, L., 1988. Fungal colonization of attached beech branches. I. Early stages of development of fungal communities. New Phytol. 110, 39–45. https://doi. org/10.1111/j.1469-8137.1988.tb00235.x.

5. Conclusions This study is the first to describe living-wood-inhabiting mycobiota of Patagonian Nothofagus species and has found that LWIF communities of N. dombeyi and N. pumilio are different from each other and are both highly diverse. For both Nothofagus species, most fungal taxa recovered were ascomycetes although a high diversity of basidiomycetes were found. Also, spatial heterogeneity across stems and roots were found, involving community composition and structure and ecological strategies of taxa present. Because it was performed with a culture-based approach, this study excluded unculturable species and, thus, likely underestimated such diversity. Now that the reference culture collection and sequence data set have been obtained, further next generation approaches using DNA directly sequenced from plant tissue will successfully complement description of living-wood-inhabiting mycobiota. As far as Nothofagus grouped mortality is concerned, health condition is a stronger driver of N. dombeyi wood mycobiota than is for N. pumilio, for which plant compartment was a stronger driver of LWIF diversity. In any case, for both Nothofagus species, symptomatic stands harbour significantly different sapwood mycobiota than apparently healthy stands. More decomposition agents and potential pathogens were found in symptomatic stands of N. dombeyi although their frequency patterns do not allow the inference of conclusions about the 11

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