Bol. Micol. 2018; 33(1):9-20 ECOLOGÍA

Diversity and growth-effects of ectomycorrhizal fungi of a in the Andes of Southern Chile (Diversidad y efectos de crecimiento de hongos ectomicorrízicos en un bosque de Nothofagus pumilio en los andes del sur de Chile)

Cesar Marín1*, Eduardo Valenzuela2, Roberto Godoy1, Götz Palfner3

1Instituto de Ciencias Ambientales y Evolutivas,Universidad Austral de Chile, Valdivia, Chile. 2Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile. 3Departamento de Botánica, Universidad de Concepción, Concepción, Chile. * Corresponding Author: [email protected]

RECIBIDO: 28 de Abril de 2018 APROBADO: 22 de Mayo de 2018

DOI: 10.22370/bolmicol.2018.33.1.1164

LOS AUTORES DECLARAN NO TENER CONFLICTO DE INTERESES

Key words: Cortinarius, ectomycorrhizal fungi, inoculation experiments, mycotrophic status, temperate rainforests. Palabras claves: bosques templados lluviosos, Cortinarius, experimentos de inoculación, estatus mico- trófico hongos ectomicorrízicos.

ABSTRACT on N. pumilio seedlings. We found that from 46 vascular identified in this study, 42 Chilean temperate rainforests have unique (91%) were mycorrhizal and of these 33 (72%) climatic, edaphic and biotic conditions, constitu- ting pre-industrial blueprint ecosystems. Myco- were associated with arbuscular mycorrhizae rrhizal associations play a central role in the (AM), two (the dominant N. pumilio and N. biogeochemical processes of these ecosystems´ dombeyi) were forming ectomycorrhizae (EM), functioning. Baseline forest ecology studies are five were associated with ericoid mycorrhizae, necessary in order to better understand diversity two with orchid mycorrhizae, and four were non- patterns, specifically regarding mycorrhizal sym- mycorrhizal. Additionally, 26 EM species were biosis. Therefore, here we describe the vegetation detected of which 15 belong to Cortinarius. Fina- characteristics and the mycorrhizal relationships lly, there were clear differences in the growth of N. of vascular in a Nothofagus pumilio forest. pumilio seedlings inoculated with the ectomycorr- We also describe, via morphological methods, the hizal fungus Laccaria laccata compared to non- ectomycorrhizal diversity present in this forest. inoculated plants. We suggest that mycorrhizal Additionally, we determined whether ectomyco- fungi play a key role in seedling colonization of rrhizal inoculation confers positive growth effects harsh environments such as the Andean treeline.

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RESUMEN of agaricoides, Valdivian and native coniferous fo- rests are largely dominated by arbuscular mycorr- Los bosques templados lluviosos de Chile hizae (AM)8,9,10,11,12,13,14,15. In EM or AM dominated tienen condiciones climáticas, edáficas y bióticas , the reciprocal mycorrhizal types (either únicas, constituyendo ecosistemas preindustria- EM or AM, respectively) are not excluded, as they les modelo. Las asociaciones micorrízicas juegan are often associated with understory plants. The un rol central en los procesos biogeoquímicos del dominant genus of EM fungi in Southern South funcionamiento de estos ecosistemas. Por lo tan- American Nothofagus forests is Cortinarius with to, en este estudio describimos las características at least 200 species described to date16,17. Boletales de la vegetación y las relaciones micorrízicas de and Russulales, which contribute to an important las plantas vasculares de un bosque de Nothofa- share of the fungal diversity in northern hemis- gus pumilio. También describimos, vía métodos phere ectotrophic forests, are scarce in Southern morfológicos, la diversidad ectomicorrízica pre- South America18,19. Nothofagus pumilio forests sente en este bosque. Adicionalmente, determina- are usually located at the vegetation limit of the mos si inoculaciones ectomicorrízicas confieren mountain region with a latitudinal extension that efectos de crecimiento positivos a semilleros de covers more than 2,000 km of distribution of this N. pumilio. Encontramos que, de 46 especies de particular forest type at the treeline of the Andes plantas vasculares identificadas en este estudio, 42 Mountains20. (91%) son micorrízicas, y de estas, 33 (72%) es- tán formando micorrizas arbusculares (AM), dos We aimed at (i) determine the vegetation (los árboles dominantes N. pumilio y N. dombe- pattern and the mycotrophic status of the vascular yi) están asociados con ectomicorrizas (EM), cin- plants, and (ii) determine the EM fungal species co están asociadas con micorrizas ericoides, dos present in a Nothofagus pumilio forest located con micorrizas orquioides, y cuatro fueron no- at the treeline of Puyehue National Park, Chile micorrizadas. Adicionalmente, 26 especies de EM (40°S). Also, we hypothesized that a native ec- fueron detectadas, de las cuales 15 pertenecen a tomycorrhizal species fosters the growth of No- Cortinarius. Finalmente, hubo claras diferencias thofagus pumilio (a dominant and test species). To en el crecimiento de los semilleros de N. pumilio test this, we (iii) measured the effects EM fungal inoculados con el hongo ectomicorrízico Laccaria inoculations on N. pumilio seedlings. laccata, comparados a plantas no inoculadas. Su- gerimos que los hongos micorrízicos juegan un rol MATERIALS AND METHODS clave en la colonización de ambientes severos por juveniles, como en el límite altitudinal andino. Study plot. We selected one 30 m x 30 m plot in the Andes INTRODUCTION Mountains of Southern Chile in Puyehue National Park (40° 47’ S – 72° 12’ W), Antillanca sector Southern Chile Nothofagus spp. forests, (near Raihuen Crater). The plot was located within which extend from central Chile to Tierra del Fue- the altitudinal limit (1,150 – 1,200 m.a.s.l.) of a go, present solely ectomycorrhizal (EM) associa- pristine, temperate forest of Nothofagus tions as fungal partners. While Nothofagaceae fo- pumilio. The plot receives more than 7,000 mm of rests exclusively form EM associations, being the annual precipitation, mostly during winter (June to only native ectomycorrhizal trees in Andean Pa- September), and the annual mean temperature is tagonian forests1,2,3,4,5,6,7 and have a high diversity 4.5°C 21.

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Vegetation patterns and mycorrhizal status. plot and selected for subsequent stratification at A previous study has shown that the crown cover 4°C. NaCl (0.1%) was applied for 30 s to aid ger- of this forest is 95%, and trees of this forest have a mination, and then a treatment with gibberellic medium height of 15 m, a maximum height of 20 acid was applied for 24 h, in order to break the m, and an approximate age of 155 years22. Daso- endogenic latency. To ensure that the inoculation metric records indicate that the density of trees in species would be compatible with the seeds co- the forest is 756 trees ha-1 with an average DBH llected, the EM fungal species Laccaria laccata of 13.3 cm and a basal area of 66.6 m2 ha-1 22. The (Scop.) Berk. was collected in the plot, transpor- floristic composition of the vascular plants in the ted to the laboratory, the fungus was isolated from plot was determined according to Marticorena & sporocarps, the mycelium grown in several petri Quezada (1985)23, and life forms were determi- dishes with malt Agar (2%) at 25°C. Following ned according to Ellenberg & Mueller-Dombois this, the fungi were cultured at 25°C in 1 L ste- (1966)24. In order to determine the types of myco- rile recipients containing peat:vermiculite (2:1) rrhizal associations, fine root samples of at least substrate and modified Melin-Norkrans (MNM) five individuals of each species were extracted medium (pH 5.0). Cultures were maintained until from soil close to the respective plant species, ve- visible development of the mycelium’s vegetative rifying the root attachment, transported to the la- growth30. There were three treatments: a control boratory, fixed in 70% alcohol, stained and obser- with natural soil and no inoculum, natural soil ved under a microscope for fungal colonization25. (from the forest plot) with inoculum, and sterile For determining ectomycorrhizal associations, soil (from the forest plot, sterilized with an auto- root samples were observed and described accor- clave at 121°C, 1 atm, for 30 minutes) with ino- ding to Agerer (1995)26 and Palfner (2001)5. culum. Sterile and pre-germinated seedlings (one seedling per pot, 20 pots per treatment, in culture Determination of ectomycorrhizal fungal spe- chambers) were selected for uniform height and a cies. 3 cm radicle; selected seedlings were then planted The EM fungal species determination was carried in 1 L pots with natural and sterile soil. out in the whole plot during periodic visits (every two months) for a period of three consecutive A 50 milliliters volume of L. laccata ino- years. Fruiting bodies of EM fungi were collected culum solid substrate was added beneath the radi- according to Agerer (1991)27 and fresh state mor- cal system of each plant. In total, 20 plants were pho-anatomic identification was conducted26,27,28. used for each of the three treatments (60 plants Following identification, the fruiting bodies were in total); this 60 pots were randomly located in dried and added to a reference collection. Taxono- the greenhouse. The plants were left to grow in a mic references of Garrido (1988)1, Valenzuela et greenhouse for 17 weeks where the soil humidity al. (1999)4, and Gamundi & Horak (1993)29 were was maintained similar to what would be found in consulted for species determination of gasteroid the field; water was supplied each week; no ferti- and agaricoid basidiomycetes, as well as ascomy- lization was implemented. At the end of the expe- cetes. rimental period, the following morphological and biomass traits were measured for all plants: root Inoculation experiment with Nothofagus pumi- collar diameter, shoot length, root length, fresh lio seedlings and ectomycorrhizal fungi. shoot weight, fresh root weight, shoot dry weight Nothofagus pumilio seeds were collected in the and root dry weight. The formation of L. laccata

Bol. Micol. 2018; 33(1):9-20 micologia.uv.cl 12 Diversity and growth-effects of ectomycorrhizal fungi - Marín C. et al mycorrhizae was confirmed in each of the inocu- orchid mycorrhizae were present in two species lated plants using a stereomicroscope. Additiona- (4%), and four plant species (9%) were non- lly, based on the above-mentioned morphological mycorrhizal (Aspleniaceae, Proteaceae, Polypo- and biomass traits, the plant Quality Index (QI)31 diaceae, and Cyperaceae). The ectomycorrhizal was calculated for the seedlings. The QI integrates and ericoid plant species were phanerophytes, several highly-correlated and non-correlated mor- while the orchid type was associated with typical phological growth traits, thus, giving a strong and herbs (cryptophytes). objective proxy of young plant quality31. Ectomycorrhizal fungi. Statistical analysis. Twenty-six EM fungi species were identified In order to test differences between the treatments in the forest plot (Table 2); most of EM species of the inoculation experiment, a Tukey test was were identified by sporocarps, except two putati- performed using the morphological traits measu- ve morphotypes which belong to the family Cor- red (p value <0.05), using the R function Tuke- ticiaceae. Cortinarius spp. were confirmed to be 32 yHSD in R . associated with N. pumilio via root-colonization analysis. Brown spored EM fungi genera (Corti- RESULTS narius and Descolea) accounted for 62% of the to- tal EM species richness, a characteristic pattern in Vegetation pattern and mycorrhizal status. Andean-Patagonian Nothofagus forests. Another Forty-six species were identified in EM genus that was found to associate with several the N. pumilio plot (Table 1). The understory ve- plants in the plot was Laccaria, which was a genus getation was mainly composed of Chusquea mon- composed of two species. Other EM fungal genera tana Phil., Drimys andina (Reiche) R.A. Rodr. et were represented by only one species (including Quez., Maytenus disticha (Hook) Urban, Berberis , Cenococcum, Gautieria, Inocybe, buxifolia Lam., Berberis serrate Lechler, Pernett- Russula, and Tricholoma). ya myrtilloides (Mirb) Blume, Pernettya pumila (L.f.) Hook and Ovidia andina (P.et E.) Meins. The herbaceous included Valeriana lapathifolia Inoculation experiment. Vahl. and Viola reichei Skottsb. We found that the inoculation of N. pumilio with the EM fungi Laccaria laccata showed a positi- Four different types of mycorrhizal asso- ve effect on seedling growth traits (Table 3). N. ciations were found for the 42 vascular plants spe- pumilio seedlings in natural soil inoculated with cies studied (91% of total vascular plant species). the fungus had clearly higher growth than the N. The AM associations were present in several life pumilio control seedlings in natural soil without forms in a total of 33 vascular plant species (72 % inoculum (Table 3). Both aboveground and be- of total vascular plant species). Two species lowground traits were significantly affected by (4 % of total vascular plant species) were asso- the fungal inoculum treatments. Plants grown in ciated with EM fungi; these included N. pumilio, natural soils with the EM fungi inoculation (Table which was the dominant tree in the plot with 85% 3) also had significantly increased growth (Table of canopy cover and N. dombeyi, which was less 3). The quality index was significantly higher in frequent with 5% of canopy cover. Ericoid myco- the inoculated treatments when compared to the rrhizae were present in five plant species (11%), control (Table 3).

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Table 1. Vascular plant species present in the Nothofagus pumilio plot, with their respective family, life form and mycotrophic status.

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Life forms: Phan=phanerophytes, Cham=chamaephytes, Cryp=cryptophytes, Hem=hemicryptophytes. Mycorrhizal status: EM=ectomycorrhiza, AM=arbuscular mycorhiza, ER=ericoid, OR=orchid, NM=non mycorrhizal. Table 2. Ectomycorrhizal (EM) fungi species identified in theNothofagus pumilio plot.

Identification by: (a)=(4); (b)=this study. “Nothofagirhiza reticulosa” described by Palfner (2001)5, “No- thofagirhiza vinicolor” described by Palfner & Godoy (1996)2.

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Table 3. Inoculation experiment, showing the growth effects of the ectomycorrhizal (EM) fungus Lacca- ria laccata in different morphological and biomass traits of Nothofagus pumilio seedlings.

Q.I.: Quality Index, resulting by combining the measured morphological traits, according to Ritchie (1984)31. *denotes statically significant differences between the control and the treatments with fungal inoculum (Tukey test, p value < 0.05).

DISCUSSION Proteacaeae (non-mycorrhizal and which has preo- teoid roots, a nutrient-uptake adaptation). In the The vascular flora found in the forest plot region, all these families are typically considered (Table 1) is typical of Chilean temperate rainfo- non-mycorrhizal9,10,11. rests located at the vegetation limit of the Andean treeline and was best classified as Drimys andina Although the study of fungal diversity has – Nothofagetum33. Other studies have documented been traditionally dependent on fruiting bodies, that this forest differs in age and growth characte- as in this study, there is an increasing necessity ristics compared to lowland temperate rainforests, of comparing these results with those from mole- likely due to the effects of frequent natural distur- cular and metagenomic studies37,38. The Puyehue bances34,35; lowland N. pumilio forests tend to be National Park, where this study took place, has older and taller than the forest described here. The been recently studied in terms of its AM fungal di- vascular flora described here has varied mycorrhi- versity14,15, and in its whole soil fungal diversity7, zal associations: Nothofagus species were found including all fungal functional groups (EM, AM, to be exclusively associated with EM fungi, while saprophytic fungi, lichens, etc.). Although these most other plant species (72%) were dominated by mentioned metagenomic studies were not conduc- AM fungi. Furthermore, ericoid mycorrhizae are ted on the exact plot as this study, we hope to give typically associated with the families Empetraceae future insights for fungal diversity comparisons and Ericaceae. Additionally, orchid mycorrhizae using traditional and modern taxonomic methods. were found in two Orchidaceae species. One spe- cies of the genus Gunnera, which is known to take Ectomycorrhizal fungi species and their ecosys- part in a facultative tripartite association (AM fun- tem roles in N. pumilio forests. gi and the N-fixing cyanobacteria Nostoc puncti- The diversity of agaricoid fungi recorded in Pata- forme36), was also found in the plot (Table 1). gonian forests is comparable to similar temperate Non-mycorrhizal plants included a representative forest ecosystems in the Northern hemisphere6 (Ta- of Cyperaceae, two species of epiphytic ferns (As- ble 2). This result is in accordance with previous pleniaceae and Polypodiaceae), and one species of studies in the region1, 2, 3, 4, 5, 6. Excluding Nothofa-

Bol. Micol. 2018; 33(1):9-20 micologia.uv.cl 16 Diversity and growth-effects of ectomycorrhizal fungi - Marín C. et al gus spp., most of the vascular flora in the region and moister soil layers, and this would be correla- is dominated by AM fungi8,9,10,11,12,13,14,15. There is ted with the formation of fruiting bodies with long, not much certainty about species-specific associa- radicating stipes during the rainy season. This is tions between the ten Chilean Nothofagaceae spe- the exact pattern of Cortinarius pugionipes, which cies and their mycobionts; it is yet unknown even has been repeatedly observed at the study region; whether there are differences in the EM commu- so far, C. pugionipes has been only observed in nity structure of and deciduous forests. N. pumilio forests16 (according to Moser & Horak However, the results here provide some evidence (1975) and the present study) and may be a spe- that Southern Andean N. pumilio forests are dis- cific mycobiont of this tree species. Likewise, the tinct, not only based on EM species composition unidentified ectomycorrhizal morphotypes (mostly but also in terms of the morphology of the encoun- dense mycelial mats under the litter) are puta- tered fungal taxa. The dominance of Cortinarius tive members (taxa) of the Corticiaceae: “Nothofa- spp. in association with N. pumilio was confirmed girhza vinicolor” and “Nothofagirhiza reticulata”. by this study. Cortinarius represented the 58% (15 These taxa so far have been found associated ex- species) of the total EM fungal richness. Most re- clusively with N. pumilio. “N. vinicolor” is easy gistered EM fungal species are common in Notho- to detect in the field by the dark red color of its fagus forests in Chile and in limitrophous zones periferical mat-like mycelium and has been obser- that span a wide latitudinal and altitudinal range in ved in several Andean N. pumilio forests and in the 1,5,39 Argentina . Examples of these common species coastal mountains of south-central Chile5. So far are Austropaxillus boletinoides, Cortinarius flam- “N. vinicolor” has not been found in association muloides, C. magellanicus, Descolea , with any other Nothofagus species. Morphological Laccaria laccata, Russsula fuegiana and Tricho- methods should be complemented with molecular loma fusipes. methods; sequencing soil samples from Argenti- nian Nothofagus forests, Marín et al, (2017a)7 and The upper soil layers in the study area con- Nouhra et al, (2013)41 found similar EM genera to sists mainly of deposits of broad pyroclastic parti- those described in this study. cles produced by repeated past volcanic eruptions. This material promotes rapid drainage of rainwater The only Ascomycete among the identified and snowmelt and, due to its dark color, heats up EM species is the cosmopolitan anamorphic spe- considerably when exposed to direct sunlight. Re- cies Cenococcum geophilum, member of the Glo- sulting water stress, which adds to highly variable niaceae family42. C. geophilum has been reported temperatures during diurnal and annual cycles, in a wide range of natural and managed forests and would likely favor basidiomycete dominance, as 3,5 these fungi form secotioid or hypogeous fruiting in greenhouse and nursery cultures . It seems to bodies (examples include Cortinarius albocanus be particularly common in soils periodically prone and Gautieria inapire). As indicated by Moser & to water stress, which reflects the conditions of our Horak (1975)16 and Horak & Moser (1965)40, the study plot. The most interesting EM species found success of secotioid ectomycorrhizae under the is probably the hypogeous basidiomycete Gautie- mentioned conditions is evident by the high diver- ria inapire. This species is so far only known from 5,43 sity of sequestrate Cortinarius species in Andean the type locality associated with N. pumilio , Patagonian Nothofagus forests. where it forms semihypogeous fruiting bodies in dense clusters. This is another striking example An alternative response to soil water stress of functional adaptation to adverse environmen- would be a retreat of the mycelia towards deeper tal conditions, as the fruiting bodies emerge from

Bol. Micol. 2018; 33(1):9-20 micologia.uv.cl 17 Diversity and growth-effects of ectomycorrhizal fungi - Marín C. et al dense mycelial mats directly beneath the soil sur- monstrating the importance and prevalence of root face. This results in moisture retention, improving symbionts in the ecology of forest ecosystems. conditions for the survival of primordia during late This study should be considered as a baseline for summer and early autumn before the rainy season further studies of mycorrhizal diversity and eco- starts. Another important role of the mentioned logy in this particular forest ecosystem. Based on mycelial mats may be the formation of a nursery identification of fruiting bodies, we listed a total bed for tree seedling establishment and survival, of 26 EM fungi species in the N. pumilio forest as has been suggested by Griffiths et al (1991)44 at the treeline of Puyehue National Park, Chile. for the closely related northern hemisphere species However, molecular methods are definitely needed Gautieria monticola. to further identify soil biota and to provide a com- plementary view of the diversity and functionali- The feedback between soil, mycorrhizae, ty of this important component of the ecosystem. and plants is especially important for plant growth The inoculation assays with EM fungi showed a at forest altitudinal limits45,46. In conservation pro- potential for using inoculations as a tool in restora- grams, if certain plants and fungi are present, there tion programs, especially in commonly disturbed is a considerable potential for N. pumilio seedling (earthquakes, fires, volcanic events) areas of the production and health (Table 3). This would have region. strong implications for a country, such as Chile, that is highly dependent on the forestry industry, ACKNOWLEDGEMENTS where sustainable local production remains to be achieved. We thank Emily Giles for her helpful com- ments regarding this manuscript. Study in memory CONCLUSIONS of Eduardo Valenzuela. This study was partia- lly funded by the CONICYT National Doctorate Most of the vascular flora (91%) in the Scholarship No. 21150047, and by the FONDE- pristine forest had mycorrhizal associations, de- CYT projects No. 1141060 and No. 3150175.

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