Mycorrhizal and Status of Obligate Facultative Epiphytic Ferns in a Valdivian Temperate Forest of Patagonia, Argentina Natalia FernAndez* and Sonia Fontenla Dep. Biologi'a General, Laboratorio de Microbiologi'a Aplicada de Biotecnologi'a, Institute y Investigaciones en Biodiversidad Medioambiente (INIBIOMA), CONICET - UNComahue, y Quintral 1250, C. de Bariloche (R8400 FRF), Negro, Argentina S. Ri'o Mari'a Ines Messuti Dep. Botanica, Institute de Investigaciones en Biodiversidad Medioambiente (INIBIOMA), y CONICET - UNComahue, Quintral 1250, S. C. de Bariloche (R8400 FRF), Rio Negro, Argentina Autotrophic epiphytes grow upon which are plants that other plants only provide physical support and are not parasitized. Plants restricted to epiphytic habitats are called "obligate epiphytes" and those that occur both epiphyti- and known cally terrestrially are as "facultative epiphytes" (Benzing, 1990; 10% Janos, 1993). Vascular epiphytes approximately constitute of vascular all and flora (Kress, 1986) play an important dynamics by role in forest contributing to species richness and by trapping and storing atmospheric making them nutrients, temporally unavailable to terrestrial plants (Benzing, 1990). * Corresponding Author: Tel.: +54 02944 428505/423374 (int.102), e-mail: natifern@yahoo. FERNANDEZ ET MYCORRHIZAL STATUS OF AL.: EPIPHYTIC FERNS Mycorrhizae generally influence plant fitness by improving nutrient uptake and increasing plant resistance to drought and other environmental stress factors (Smith and Read, 1997; Read, 1999). Despite the importance and abundance known of epiphytes, about mycorrhizal little is associations in these (Bermudes and plants Benzing, Allen 1989; etal., 1993; Janos, 1993; Michelsen, Epiphytic 1993). habitats are usually considered extreme environments for plants because they often are subjected to great variation in temperature and in water and nutrient availability (Benzing, 1990). Therefore, generally is it may expected that mycorrhizae be an important adaptation epiphytes for (Benzing, 1990; Lesica and Antibus, 1990; Janos, 1993; Rains et al, 2003). Nevertheless, has been observed it that epiphytic species are usually non- mycorrhizal (Allen (commonly et al, 1993), facultative mycorrhizal mycorrhizal when grow they and when terrestrially inconsistently mycorrhizal they grow epiphytically) (Maffia et al, 1993; Michelsen, 1993; Nadarajah and Nawawi, and when AM, 1993), that they have colonization usually low (Bermudes and is Benzing, and Rowe 1989; Lesica Antibus, and 1990; Pringle, 2005). Studies concerning the occurrence of mycorrhizae in epiphytes are few and only the Orchidaceae and Ericaceae have been families relatively well studied (Bermudes and Benzing, and 1989; Lesica Antibus, 1990; Dearnaley, 2007). Some species included in the Bromeliaceae (Allen 1993; Rabatin et al., et al, Rowe and 1993; Pringle, 2005) and other predominant famiHes of vascular epiphytes (such as the Araceae, Cyclanthaceae and Piperaceae) have been also analyzed (Bermudes and Benzing, and 1989; Lesica Antibus, 1990; Janos, 1993; Maffia et al., 1993; Michelsen, 1993). However, the mycorrhizal status of epiphytic species included in families other than those mentioned here remains an open much more question. Therefore, information needed before is generalizations about mycorrhizae in epiphytic environments can be made (Bermudes and Benzing, 1989; Allen et al, 1993; Janos, 1993). Abundant among epiphytes are present the vegetation of the Valdivian temperate forests of South America. Within these forests, seedless vascular mostly plants, ferns (monilophytes, see Pryer 2004 and Smith et al., et al., 2006), one most are of the important components The of the epiphytic vascular flora. mycorrhizal group status of this of plants poorly understood (Dhillion, 1993; is much Zhao, and known 2000) about symbiosis less is this in epiphytic species. This research was conducted where in Puerto Blest, previous studies have AM demonstrated the presence of in different plant species (Chaia et 2006), al., HBK including Equisetum bogotense and Lycopodium paniculatum Desv. ex two Poir., species of seedless vascular plants which were found be to facultative mycorrhizal (Fernandez The aim et 2008). of the present study al., AM was to analyze the occurrence of in sporophytes of different obligate and facultative epiphytic fern species. Materials and Methods Study area.—The Valdivian temperate South America forests of are included in the Subantarctic Province (Subantarctic Domain, Antarctic Region). This AMERICAN FERN VOLUME NUMBER JOURNAL: 100 1 (2010) / \ (ieographical location •ic. 1 of tl . jointed out in a black rectangle, L ecoregion located in the Southern region of Argentina and Chile, from 35° is to 48 S latitude between the Eastern slope of the Andes and the Pacific Ocean. W) Puerto Blest (4r 02' S, 71 49' part of the Valdivian temperate forest is region of Argentina and situated within the Nahuel Huapi National Park in it is Negro Province, Patagonia Armesto Ri'o (Cabrera, 1976; et aL, 1995) (Fig. 1). It one is of the rainiest places in the country, with an average annual rainfall of mm 3000 and an annual average temperature 9^ The dominant of C. tree species hydrophilic Nothofagus dombeyi some in this forest (Mirb.) Oerst. In is mixed IM areas, this species forms forests with Fitzroya cupressoides (Molina) Johnst., Saxegothaea conspicua Lindl., Dasyphyllum diacanthoides (Less.) and Schodde Cabrera, Laureliopsis philippiana (Looser) (Dimitri, 1972; Brion et ai, 1988; Correa, 1998). Trunks and branches of these tree species are usually covered by different epiphytes, including lichens, mosses and several ferns. Epiphytic ferns present in Puerto Blest are small plants that range in size from 20 cm. 2 to — Sampling. Five samplings were conducted Puerto between in Blest spring 2004 and autumn 2006. Sporophytes of epiphytic ferns were sampled by random walk from trunks and branches of different tree species, such as N. dombeyi, S. conspicua, D. diacanthoides and philippiana, which were L. m away The situated at least 5 from the footpath. epiph5^ic specimens were m collected from 0.5 to 2 height above the ground. Species that were found to be also growing on the forest soil were considered as facultative epiphytes and root samples were collected from epiphytic as well as from the conspecific FERNANDEZ ET MYCORRHIZAL STATUS OF AL.: EPIPHYTIC FERNS One specimens. Hymenophyllum terrestrial individual of dentatum, a species that is reported to be epiphytic (Rodriguez, 1995) but was found only on the was ground, also sampled. Sporophytes were removed carefully from the substrate in order to preserve most of the root system and were stored in labeled plastic bags and refrigerated 4°C at until processed. Roots from samples were separated from all the rest of washed and the plant, carefully cleaned under a stereoscopic microscope (Olympus SZ remove 30) so as to all root pieces belonging to other plants. Samples were 70% fixed in formalin-acetic acid-alcohol (FAA). Analyses samples.— of the All samples were stained using modified a Hayman and method Phillips Fernandez (1970) (see et al, 2008). For each individual three slides were made, each of them with ten stained root pieces of cm approximately mounted 1 length in glycerol. Root pieces were examined with a light microscope (Olympus BX40) 20 X magnification. The at criterion AM used in this study for the determination of was the presence of arbuscules AM one in at least individual of each species. Structures corresponding to or other root fungi were documented as brightfield images, which were captured with a camera (Sony ExwaveHAD) and digital Image-Pro Plus 4.1.0.0. analysis Windows. software for Results A of 66 sporophytes belonging total to ten species in three families were Ten sampled. specimens (15%) corresponded Asplenium to dareoides, eight (12%) to Grammitis magellanica and 48 (73%) to various species of the family Hymenophyllaceae. In Puerto Blest, A. dareoides and G. magellanica are the only representatives of the Aspleniaceae and Polypodiaceae families respec- tively (Brion et al, 1988; de la Sota et al., 1998). At least five sporophytes were found most for of the species, except for H. cuneatum, H. falklandicum and H. which ferrugineum, were represented by only one two specimens or (Table 1). Most of the samples (73%) were collected from epiphytic and we habitats observed that they frequently had attached soil particles to the outside of the probably consequence roots, as a of their proximity to the ground. Terrestrial sporophytes (27%) were from collected loose organic with abundant soils leaf wood and them litter, 2 (11%) of belonged to A. dareoides and 16 (89%) to five Hymenophyllum species of (Table All the sporophytes examined presented 1). mm extensive, thin (less than 2 wide) and fibrous root systems with abundant and root hairs. Baylis (1975) John described system St. (1980) this root morphology "graminoid". as AM Most of the sporophytes lacked structures, but in seven terrestrial samples (11% of the total specimens analyzed), corresponding H. to pectinatum and H. seselifolium (Table scarce knobby hyphae and 1), 5% structures similar to coils and vesicles were observed (less than of the root length examined) (Fig. However, arbuscules were not detected in any 2). of these specimens, so questionable these plants can be designated it is if as mycorrhizal. AMERICAN FERN VOLUME NUMBER JOURNAL: 100 1 (2010) Abundant and hyphae and extra- intracellular of regularly septate generally melanized fungi frequently associated with microsclerotia and "cerebriform were They structures" present in root systems corresponded all (Fig. to 3]. named dematiaceous endophytes root usually dark septate fungi (DSF). Discussion Of examined the ten species in the present study, G. magellanica and S. caespitosa were found to be obligate epiphytes (only growing as epiphytes), while A. dareoides and five species of Hymenophyllum [H. cuneatum, H. pectinatum, H. seselifolium, H. tortuosum and H. umbratile] were facultative epiphytes (growing These either epiphytically or terrestrially). observations who agree with de la Sota et (1998), described the same habits for these al. We species. considered H. cuneatum as facultative epiphytic even though only one terrestrial sporophyte was collected because described as epiphytic in is it Hymenophyllum the literature (Rodriguez, 1995). falklandicum and H. ferrugineum could not be classified as obligate or facultative epiphytes because only one or two individuals per species were found (Table 1). Representatives of the Aspleniaceae, Polypodiaceae and most of the AM Hymenophyllaceae families in Puerto Blest clearly lack colonization. Chaia and Fernandez observed et al. (2006) et al. (2008) that different plant AM. species growing in areas adjacent our study had Furthermore, to site AM Chaia found et al. (2006) out that in Puerto Blest the soil infective capacity ranges from lU These 0.6 to 1.7 g/soil. findings indirectly demonstrate that FERNANDEZ ET AL.: MYCORRHIZAL STATUS OF EPIPHYTIC FERNS Fungal colonization Fig. 2. similar to arlniscular nivcoirhizas in //. pectinatum (a,b) and H. seselitnhum and (c,fl). a: Extra intracelhilar mycelia wliorc soine sopta can be observed, b,c: AM inoculum where there in the the samples were However, is soil collected. specimens terrestrial of A. dareoides, H. cuneatum, H. tortuosum and H. AM umhratile did not form despite the presence of inoculum in the which soil, supports the non-mycorrhizal behavior of these species. Even though we found structures similar to vesicles and coils in roots of four out of five terrestrial sporophytes of H. pectinatum and in three out of seven sporophytes terrestrial of H. seselifolium (Fig. was not possible to record them as mycorrhizal 2), it because arbuscules (the diagnostic feature of the symbiosis) were absent and the colonization percentage was very low than 5%). Additionally, (less is it known some that other fungi are capable of forming structures similar those to AM within some Rowe of roots of epiphytic plants (Brundrett, 2002; and These Pringle, 2005]. structures can also stain dark blue (Fig. 2c,d) and in AM may consequence they be mistaken The for (Janos, 1993). presence of such fungi could explain the presence of this type of structures in roots of H. pectinatum and H. seselifolium. The absence of mycorrhizae in the epiphytic fern species examined in this work is concordant with previous studies that have analyzed this symbiosis in Gemma other species belonging to the same families (Cooper, 1977; et al, Wang 1992; Zhao, 2000; and Qiu, but from some 2006), differs others have that AMERICAN VOLUME NUMBER FERN [OURNAL: 22 100 (2010) 1 Dark Fk;. 3. soptato lungi in roots of opipinytic: t<iriis of Puerto Blest, a: Extraradical hypha and intracelkilar microsclerotia in S. caespitosa, b: General view of extraradical DSF colonization in G. ^ a=50 Scale ^m; b,c=100 bars: d,e,f=20 \im; urn. AM reported the occurrence same group of in the of plants (Cooper, 1976; Iqbal Gemma Muthukumar and et al, 1981; et 1992; Udaiyan, 2000; Zhao, 2000; al., Wang and Our Qiu, 2006). results are also consistent with Lesica and Antibus who and Michelsen examined and (1990) (1993), 13 eight species of epiphytic ferns in Costa Rica and Ethiopia respectively (including five species of Asplenium and one Hymenophyllum] and them of described non- as , Nawawi mycorrhizal. Nadarajah and (1993) studied the mycorrhizal status of They 19 fern species in Malaysia. observed that these species were non- all AM mycorrhizal in epiphytic habitats and that eight of them were able form to when and/or were vesicles coils they rooted in Nevertheless, soil. as it occurred in this work, these authors did not detect arbuscules in any of the AM Although has been suggested by several authors that are generally it absent (Allen et al, 1993) or rare (Bermudes and Benzing, 1989; Lesica and Antibus, 1990; Maffia et al, 1993, 2000; Michelsen, 1993; Nadarajah and Nawawi, 1993) in epiphytes. Rains et (2003) found abundant mycorrhizal al. epiphytes in a Costa Rican cloud forest. This apparent discrepancy suggests and that the occurrence, possibly the role, of mycorrhizae in epiphytic habitats may is variable and be related to taxonomic affinities and diverse ecological and factors (Tester et 1987; Lesica Antibus, 1990; Michelsen, 1993). al., AM Different explanations have been suggested for the paucity of in epiphytic For example, might be environments habitats. that these are too it dry and exposed support fungus to the or that the rate of photosynthesis FERNANDEZ ET MYCORRHIZAL STATUS OF AL.: EPIPHYTIC FERNS (which low may is relatively in epiphytes, see Benzing, 1986) be insufficient to maintain the symbiosis (Lesica and Antibus, 1990). Nutritional insufficiency, and consequently the inability to sustain the energy cost of mycorrhizae, has been also suggested as a major factor determining mycorrhizal success in epiphytes (Benzing, Some 1990; Janos, 1993). substrate analyses have demonstrated that trunks and canopy mats might be (Bermudes nutrient rich and Benzing, 1989; Lesica and Antibus, and known 1990), high that it is AM suppresses fertility of facultatively mycotrophic species (Janos, 2007). AM Another widespread explanation is that epiphytic habitats are deficient in inocula, so epiphytes usually do not have the opportunity of being colonized by mycorrhizal and fungi (Lesica Antibus, 1990; Janos, 1993; Michelsen, 1993). This suggestion contrasts with several authors (Mcllveen and Cole, 1976; Rabatin Mangan who et ah, 1993; Janos et al, 1995; and Adler, 2000) have AM may reported that spores and mycelia be dispersed epiphytic to environments by several vectors mainly and mammals). birds, ants small (e.g., AM Moreover, other studies have established that many epiphytes lack despite the presence of spores and external mycelia in the substrate (Lesica and Antibus, 1990; Maffia et al, 1993; Rabatin et ah, 1993). was In study compare this it possible to the mycorrhizal status of six facultative epiphytic fern species. In the case oi A. dareoides, H. cuneatum, H. tortuosum and AM, H. umbratile specimens lacked and most all in of the terrestrial sporophytes of H. pectinatum (73%) and H. seselifolium (73%) AM fungal structures similar to those of were completely absent. Therefore, AM deficiencies in inocula or in nutrient acquisition (which not are limiting when factors plants are rooted in as has been demonstrated previous soil, in studies carried out Puerto would in Blest) not explain the non-mycorrhizal behavior observed in these plants. Thus, is necessary to seek other it explanations phenomenon. Another for this factor that might explain the AM absence of in epiphytic ferns of Puerto Blest root system morphology. is Plants with long root length (Koide and 1991; Ryser and Lambers, Li, 1995), small root diameter (Reinhardt and Manjunath and Miller, 1990; Habte, 1991; Hetrick and abundant et al., 1992) long root hairs (Baylis, 1975; John, 1980; St. Schweiger et al, 1995), like those observed in this study ("graminoid roots"), tend to be non-mycorrhizal (Baylis, 1975; John, 1980; Hetrick, 1991; St. Michelsen, Zangaro 1993; et 2005). al., Dark septate fungi (DSF) are conidial or sterile fungi that have been reported in several plant species from (Jumpponen and different habitats Trappe, 1998; Jumpponen, 2001). Characteristic and hyphae and inter- intracellular micro- DSF sclerotia corresponding to were observed in every specimen analyzed in work this (Fig. 3). This is in accordance with previous studies that have reported the existence of this type of fungi within the roots of angiosperm epiphytes (Bermudes and Benzing, 1989; Allen et 1993; Janos, 1993; Rains al., and et al., 1993) several fern species (Cooper, 1976; Dhillion, 1993; Fernandez The presence et al., 2008). of "cerebriform microsclerotia" (Fig. has not 2f) been described in the literature before, except in bogotense and E. L. paniculatum, two facultative mycorrhizal species also studied in Puerto Blest AMERICAN FERN JOURNAL: The DSF (Fernandez et al, 2008). presence of within the roots of different fern species demonstrates that these plants are capable of forming plant-fungal associations with a diversity of fungal lineages (Winther and Friedman, 2007). AM According to Allen et al. (1993) and Rains et al. (2003), the lack of in most DSF many of the epiphytes in addition to the occurrence of in of them is and may DSF may notable suggest that function as "mycorrhizal fungi", but study needed further hypothesis. to test this is This study provides novel information about the occurrence of mycorrhizae and work which in epiphytic ferns the in the mycorrhizal status of first it epiphytic species of Patagonia, Argentina recorded. More work on a larger is needed scale not only for understanding the behavior of epiphytic species of is Puerto Blest but also for developing patterns of mycorrhizal activity in and epiphytic habitats for determining ecological explanations for the paucity AM and of in these plants (Lesica Antibus, 1990; Allen et 1993; Nadarajah al., and Nawawi, 1993). FERNANDEZ ET AL.: MYCORRHIZAL STATUS OF EPIPHYTIC FERNS