Arbuscular mycorrhizas and dark septate fungi in Lycopodium paniculatum (Lycopodiaceae) and Equisetum bogotense (Equisetaceae) in a Valdivian temperate forest of Patagonia, Argentina.

ABSTRACT.--Arbuscular mycorrhizas (AM) are one of the most widespread and common type of symbiotic associations. Lycopodium paniculatum and Equisetum bogotense are two important species of seedless vascular plants in a Valdivian temperate forest of Patagonia, Argentina. The mycorrhizal status of these species is completely unknown, as it is for most lycophytes and monilophytes in Argentina, where information on symbiotic interactions in these plants is scarce. In this study, typical AM structures were observed in sporophytes of L. paniculatum and E. bogotense. The percentage of root length colonized by AM fungi ranged from 0 to 50% in the first species and from 0 to 22.5% in E. bogotense. Both species were facultative mycorrhizal and it was observed that the habitat and substrate seem to play an important role in determining the colonization intensity. The morphological AM colonization pattern was considered an Intermediate-type. Dark septate fungi, characterized by septate hyphae and microsclerotia, were also present within the roots of L. paniculatum and E. bogotense.

KEY WORDS.--arbuscular mycorrhiza, morphological type, Intermediate-type, dark septate fungi, Lycopodium, Equisetum, Valdivian temperate forest

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Mycorrhizas are symbiotic associations, usually mutualistic, that improve plant fitness and influence plant biodiversity and productivity in natural ecosystems (Brundrett, 1991; Read, 1991; Smith and Read, 1997). Arbuscular mycorrhizas (AM), as the most widespread type of mycorrhizas, form symbiotic interactions with the roots of ~80% of all terrestrial plant species (van der Heijden et al., 1998; Read, 1999). There are two major morphological types of AM: Arum and Paris-type. In the Arum-type the fungi form extensive intercellular hyphae in the root cortex and then develop short lateral branches into cortical cells forming arbuscules as terminal structures on trunk hyphae. In contrast, in the Paris-type intercellular hyphae are lacking and the fungi spread directly from cell to cell forming extensive coils frequently developing intercalary arbuscules (structures known as "arbusculate-coils") (Smith and Smith, 1997).

Seedless vascular plants (including extant lycophytes and monilophytes (Kranz and Huss, 1996; Pryer et al., 2001, 2004; Smith et al., 2006)) are of ancient origin and occupy a very important position in the origin and evolution of vascular plants (Remy et al., 1994). Arbuscular mycorrhizas have been found in some fossil seedless vascular plants, and in spite of having persisted for 400 million years (MY) (Pirozynski and Malloch, 1975; Malloch, 1987; Hass et al., 1994; Remy et al., 1994), the AM status of extant lycophytes and monilophytes is poorly understood (Dhillion, 1993; Zhao, 2000; Zhang et al., 2004).

Some authors (Boullard, 1957; Hepden, 1960; Freeberg, 1962; Cooper, 1976; Iqbal et al., 1981; Berch and Kendrick, 1982; Koske et al., 1985; Harley and Harley, 1987; Duckett and Ligrone, 1992; Gemma et al., 1992; Schmid and Oberwinkler, 1993, 1994; Swatzell et al., 1996; Treu et al., 1996; Turnau et al., 2005; Muthukumar and Udaiyan, 2000; Zhao, 2000 and Zhang et al., 2004; Winther and Friedman, 2007) have studied the occurrence of AM interactions in sporophytes and gametophytes of different seedless vascular plants, including several Lycopodium and Equisetum species. According to Read et al. (2000) and Winther and Friedman (2007), Lycopodium gamethophytes appear to be obligatory mycotrophic, but there is little consensus regarding the AM associations in the sporophytes. On the other hand, Read et al. (2000) point out that those surveys concerning the mycorrhizal status of different Equisetum species have shown that the gametophytic generation seems to be non-mycotrophic and that the sporophytes could be non-mycorrhizal as well as capable of forming AM. From this analysis, it becomes evident that a significant discrepancy exists in our knowledge of the occurrence of fungal symbioses in Lycopodium and Equisetum species.

In addition to AM, another type of root colonizing fungi, called dark septate fungi (DSF), has been also reported within the roots of different seedless vascular plants (Cooper, 1976; Berch and Kendrick, 1982; Dhillion, 1993; Jumpponen and Trappe, 1998). Dark septate fungi are defined by Jumpponen (2001) as conidial or sterile fungi that colonize living plant roots without causing any apparent negative effects. The ecology, taxonomic affinities and host range of these DSF are largely unknown, as is their influence on the host and plant communities (Peterson et al., 2004). Including DSF in mycorrhizal studies would yield valuable information about the importance and frequency of these root colonizers (Jumpponen and Trappe, 1998).

Seedless vascular plants are abundant among the vegetation of the Valdivian temperate forests of South America, which are characterized by their levels of endemism and the antiquity of their ecological interactions. This ecoregion is located in the Southern area of Argentina and Chile, running from 35[degrees] to 48[degrees] S latitude, between the Eastern slope of the Andes and the Pacific Ocean (Cabrera, 1976; Armesto et al., 1995). This study was carried out in Puerto Blest, which is situated in the Valdivian temperate forest region of Argentina. Two important seedless vascular plants species present in this region are Lycopodium paniculatum Desv. ex Poir. and Equisetum bogotense HBK. The first species is endemic to the temperate forest of Argentina and Chile (between 38 and 48 S latitude), while E. bogotense has a wider geographical distribution and is restricted to moist habitats (close to streams, rivers and lake coasts) of the Andean mountain region (Correa, 1998). Currently, the mycorrhizal status of these two species it is completely unknown.

The aim of the present study was to analyze the occurrence and abundance of mycorrhizal symbionts in sporophytes of the endemic L. paniculatum and the more widely distributed E. bogotense in Puerto Blest, a Valdivian temperate forest of Patagonia. Lycopodium paniculatum and E. bogotense are the only species of each genus growing in this Valdivian temperate forest, and they are relatively rare compared to other non-seed plant species. A common feature between these species that distinguish them from the rest of the seedless vascular plants of Puerto Blest is the presence of microphylls (Brion et al., 1988; Gifford and Foster, 1989; Sitte et al., 2004). Lycopodium paniculatum and E. bogotense specimens were sampled from the forest dominated by Nothofagus dombeyi (Mirb.) Oerst. and a waterlogged peat bog because these are the most important vegetation units of this Valdivian temperate forest.

MATERIALS AND METHODS

Study Area.--This study was carried out in Puerto Blest (41[degrees] 02' S; 71[degrees] 49' W), within the Nahuel Huapi National Park in Rio Negro Province, Patagonia, Argentina. Puerto Blest is part of the Valdivian temperate forest region of Argentina. It is one of the rainiest places in the country and its average annual rainfall is 3000 mm. The annual average temperature is 9 C (Dimitri, 1972; Brion et al., 1988). In this hydrophilic forest, N. dombeyi is the dominant tree species. However, in some areas it forms mixed forests with Fitzroya cupressoides (Molina) I. M. Johnst., Saxegothaea conspicua Lindl., Dasyphyllure diacanthoides (Less.) Cabrera and Laureliopsis philippiana (Looser) Schodde. Species such as Lomatia ferruginea (Cav.) R. Br. and L. hirsuta (Lam.) Diels ex J. F. Macbr., Desfontainea spinosa Ruiz and Pav., Azara lanceolata Hook. f., Berberis darwinii Hook., Fuchsia magellanica Lam. and Chusquea culeou Desv. are very common in the shrub-layer. Within Puerto Blest forest there is a waterlogged peat bog, which corresponds to a water-retaining Sphagnum magellanicum Brid. prairie where F. cupressoides and Pilgerodendron uviferum (D. Don) Florin are the dominant tree species (Roig, 1998).

Sampling procedures.--Sporophytes of L. paniculatum and E. bogotense were collected in autumn of 2006 in Puerto Blest. The forest dominated by N. dombeyi and the waterlogged peat bog were chosen as sampling environments because they are the most representative vegetation units of this Valdivian temperate forest.

Specimens were collected from terrestrial and epiphytic habitats such as: soil in the forest, soil at the edge of the road, soil of the coast of the lake and trunk surfaces. Due to the presence of a road, soil is usually removed and there were two different conditions on its edges: stony and sandy soil; it was observed that the last one usually has higher soil moisture than the former. Sporophytes were sampled by random walk and were carefully removed from the substrate with a shovel. Each specimen was stored in a labelled plastic bag and refrigerated at 4[degrees] C until further procedures. Afterwards, roots from all samples were first excised from the rest of the plant, and then carefully rinsed with tap water, cleaned under a stereoscopic microscope (Olympus SZ 30) and fixed in 70% formalin-acetic acid-alcohol (FAA).

Clearing and staining.--All samples were stained using a modified Phillips and Hayman (1970) method. Roots were first cleared with 10% (w/v) KOH at 98[degrees] C for 1 h. After clearing, L. paniculatum roots were bleached with a 5% (v/ v) [H.sub.2][O.sub.2] (30V) and 0.5% N[H.sub.3] solution for 10 minutes. Equisetum bogotense roots were darker, so they were bleached in this solution for 40 minutes. All samples were acidified with 1% v/v HCl for 5 minutes and stained with Trypan Blue in acidic glycerol (31% v/v glycerol, 31% v/v lactic acid and 0.05% w/v Trypan Blue) by heating them at 98[degrees] C for 15 minutes. The stained roots were stored in acidic glycerol.

Analyses of the samples.--For each specimen, ten stained root pieces approximately 1 cm long were mounted on a slide in Glycerol and were examined with a light microscope (Olympus BX40). A total of three replicates of each individual were made. Typical structures that indicated the presence of mycorrhizas or other root associated fungi, such as DSF, were documented as brightfield images, which were captured with a digital camera (Sony ExwaveHAD) and Image-Pro Plus 4.1.0.0. analysis software for Windows.

The mycorrhizal type present in each sample was designated according to Harley and Smith (1983) classification. If one of the species was found to be mycorrhizal in one habitat but not in others, it was scored as facultative mycorrhizae. The criteria used in this study for the determination of AM was the presence of arbuscules at least in one individual of each species and the occurrence of the rest of typical AM structures in the samples.

Characteristic AM structures (intra or intercellular hyphae, vesicles, coils and arbuscules) were used for their classification into Arum or Paris-type (Smith and Smith, 1997). The percentage of root length colonized by AM fungi was estimated according to the magnified line-intersect method described by McGonigle et al. (1990).

RESULTS

A total of 14 sporophytes of L. paniculatum, 12 of them terrestrial and the other two epiphytic, and 12 terrestrial sporophytes of E. bogotense were collected. Typical AM fungal structures were observed in six specimens of L. paniculatum (43%) and in eight specimens of E. bogotense (67%). All colonized sporophytes had knobby hyphae (which were in general coenocytic), vesicles (Fig. 1a,d) and some coils characteristic of AM fungi (Fig. 1b,e). Arbuscules were found in four of six (67%) and in six of eight (75%) colonized specimens of L. paniculatum (Fig. 1c) and E. bogotense (Fig. lf), respectively (Table 1).

[FIGURE 1 OMITTED]

The percentage of root length colonized by AM fungi varied widely within each species. In L. panieulatum these values ranged from 0 to 50% with a mean value of 9.5%. Arbuscular mycorrhizas were observed in six of 12 terrestrial sporophytes and epiphytic individuals were not infected. The specimen with the highest value of root length colonized by AM was collected in the forest (50%); however this vegetation unit presented a lower proportion of colonized individuals than the waterlogged peat bog. The specimen that showed the lowest percentage of root length colonized by AM fungi (2%) was found in the waterlogged peat bog (Table 1).

In E. bogotense the percentage of root length colonized by AM fungi ranged between 0 and 22.5%, the mean value was 8.6%. All the individuals colonized by AM fungi were collected from the stony edge of the road. Those specimens sampled from sandy soil at the edge of the road and in the coast of the lake were not infected (Table 1).

The AM colonization observed in these two species could not be clearly classified as Arum or Paris-type. Some intracellular coils were seen in L. paniculatum and E. bogotense (Fig. 1b,e) and the first species also presented some well-defined, cell-to-cell hyphae. Only one arbuscule within each cell was observed (Fig. 1c, f) and intercalary arbuscules ("arbusculate-coils") were not detected, as would be expected in Paris-type pattern. In addition, both species lacked typical Arum-type intercellular hyphae. Therefore, the morphological AM type observed in L. paniculatum and E. bogotense was considered as an Intermediate-type.

Other endophytic fungi were present in all the samples of both species. Most distinctive structures were dark septate intra and extracellular hyphae, microsclerotia and cerebriform structures (Fig. 2), which corresponded to DSF.

DISCUSSION

In this study, it was observed that L. paniculatum and E. bogotense collected at Puerto Blest were both capable of forming AM. The existence of mycorrhizal as well as non-mycorrhizal individuals within each species supports their facultative behavior, a phenomenon that has been observed in other Lycopodium and Equisetum species in different regions of the world. For example, L. japonicum Thunb. was documented by Zhao (2000) as facultative mycotrophic in China, while Muthukumar and Udaiyan (2000) recorded the same species as non-mycorrhizal in India. In the case of E. arvense L., it had AM in Norway and USA (Dhillion, 1993), was described as facultative mycorrhizal in Canada (Berch and Kendrick, 1982) and was considered as non-mycorrhizal in Alaska (Treu et al., 1996). A similar discrepancy has been observed in L. clavatum L. and in E. hyemale L., E. scirpoides Michx. and E. variegatum Schleich. (Berch and Kendrick, 1982; Koske et al., 1985; Harley and Harley, 1987; Dhillion, 1993; Treu et al., 1996; Winther and Friedman, 2007). This information is in agreement with Boullard (1957), who carried out one of the most extensive surveys about the mycorrhizal status of seedless vascular plants and established that the mycorrhizal symbiosis within this group ranges from obligate to facultative or non-mycorrhizal. It also accords with the results obtained by Zhao (2000), who studied the mycorrhizal status of 256 species of lycophytes and monilophytes and found out that 16% of them were facultative mycorrhizal.

[FIGURE 2 OMITTED]

Although arbuscules are ephemeral structures which may be difficult to see in field-collected roots (Brundrett, 2004), they were found in many sporophytes of both species. The presence of vesicles, hyphal coils and intraradical non-septate hyphae, which are also considered as features of AM colonization by other authors (Duckett and Ligrone, 1992; Dhillion, 1993; Smith and Read, 1997; Zhang et al., 2004), allowed the detection and quantification of AM fungi in samples where arbuscules were not observed.

The colonized specimens of L. paniculatum and E. bogotense collected in Puerto Blest presented an Intermediate-type of AM colonization. This morphological type has been described for other plants (Dickson, 2004), including some other species of lycophytes and monilophytes (Duckett and Ligrone, 1991; Zhang et al., 2004). Within the Lycophyta, Zhang et al. (2004) found an Intermediate-type pattern in Selaginella moellendorfii Hieron. However, the same authors observed only Paris-type AM among the Monilophyta, including two species of Equisetum (E. hyemale and E. ramosissimum Desf.), and Winther and Friedman (2007) cited the same AM morphological type for L. clavatum. Therefore, these genera seem to be able to form both Paris and Intermediate-types of mycorrhizal symbioses.

Although this survey does not allow definitive conclusions about the relationship between AM colonization and habitats or substrates, it was observed that L. paniculatum showed a higher number of colonized individuals in the waterlogged peat bog than in the forest. Equisetum bogotense had a different symbiotic behavior depending on the type of soil (substrate) where the samples were found; AM were present in specimens growing in stony soil but were absent in those plants collected from moist sandy soil from the edge of the road and from the coast of the lake. A similar trend was previously described by Koske et al. (1985) and Dhillion (1993) in different species of Equisetum. They suggested that the absence of mycorrhizas in moist sites may be more indicative of the effect of high soil moisture than of the mycotrophic potential of this genus. Therefore, as was observed by Gemma et al. (1992) among the seedless vascular plants of Hawaii, the habitat and substrate seem to play an important role in determining the colonization intensity of L. paniculatum and E. bogotense, but this fact needs to be further studied.

Dark septate fungi were present in every plant of L. paniculatum and E. bogotense analyzed in this work. These results agree with previous studies that have recorded the occurrence of DSF within the roots of different seedless vascular plant species (Cooper, 1976; Berch and Kendrick, 1982; Dhillion, 1993; Jumpponen and Trappe, 1998), but differ from the results obtained by Winther and Friedman (2007), who did not find any evidence of nonglomalean AM associations in different species included in Lycopodiaceae. The occurrence of cerebriform microsclerotia in L. paniculatum and E. bogotense is especially remarkable, because these structures have not been previously cited in the literature. As it is becoming important to report and study DSF in order to stress some common features among these fungi and to understand how they influence the host and their relationship with AM fungi, the occurrence of DSF within the roots of these plant species is novel and relevant and supports the idea that some seedless vascular plant are capable of forming plant-fungal associations with a diversity of fungal lineages (Winther and Friedman, 2007). This information would be very useful to elucidate the nature and ecological importance of these poorly known root colonizing fungi (Jumpponen and Trappe, 1998; Jumpponen, 2001; Peterson et al., 2004).

Sporophytes of Equicetaceae and Lycopodiaceae have been regarded as non-mycorrhizal by several authors, however, the results in this study and previous reports indicate that plants of both families may be extensively colonized by AM fungi. Lycopodium paniculatum and E. bogotense collected in this Valdivian temperate forest of Patagonia are not obligate mycorrhizal species, but they develop AM under certain conditions, probably related to the habitat and the substrate where they are growing. In spite of the fact that Paris-type is the most common type of AM among the seedless vascular plants (Smith and Smith, 1997; Zhang et al., 2004), the sporophytes of L. paniculatum and E. bogotense were found to be colonized by an Intermediate-type of AM. This work represents the first record of AM fungi in lycophytes and monilophytes of the Valdivian rainforest, and constitutes the initial step in the study of the importance of AM fungi and DSF in these groups of plants.

ACKNOWLEDGEMENTS

The authors thank Dr. R. Larry Peterson (University of Guelph, Canada) and Dr. Daniel E. Martinez (Pomona Collage, USA) for the correction of the manuscript and their helpful suggestions. We are grateful to Parques Nacionales for giving us the possibility of working in a protected area. Funds for this research were provided by grant B-121 (Universidad Nacional del Comahue) and PIP 5270 (CONICET).

LITERATURE CITED

ARMESTO, J. J., P. LEON-LOBOS and M. K. ARROYO. 1995. Los bosques templados del sur de Chile y Argentina: Una isla biogeografica. Pp. 23-28, In: Armesto, J. J., c. Villagran and M. K. Arroyo, eds. Ecologia de los bosques nativos de Chile. Editorial Universitaria Santiago de Chile, Chile.

BERCH, S. M. and B. KENDRIK. 1982. Vesicular arbuscular mycorrhizae of southern Ontario ferns and fern allies. Mycologia 74:769-776.

BOULLARD, B. 1957. La mycotrophie chez les Pteridophyte. Sa frequence. Ses caracteres. Sa signification. Botaniste 41:5-15.

BRION, C., J. PUNTIERI, D. GRIGERA and S. CALVELO. 1988. Flora de Puerto Blest y sus alrededores. Universidad Nacional del Comahue, Centro Regional Universitario Bariloche, General Roca.

BRUNDRETT, M. 1991. Mycorrhizas in natural ecosystems. Adv. Ecol. Res. 21:171-313.

BRUNDRETT, M. 2004. Diversity and classification of mycorrhizal associations. Biol. Rev. 79:473-495.

CABRERA, A. L. 1976. Territorios fitogeogrdficos de la Republica Argentina. Enc Argent Agricul y Jard, Editorial Acme SACI, Buenos Aires.

COOPER, K. M. 1976. A field survey of mycorrhizas in New Zealand ferns. New Zealand J. Bot 14:169-181.

CORREA, M. N. 1998. Flora Patagonica. Parte I. INTA, Buenos Aires.

DHILLION, S. S. 1993. Vesicular-arbuscular mycorrhizae of Equisetum species in Norway and USA.: occurrence and mycotrophy. Mycol. Res. 97:656-660.

DICKSON, S. 2004. The Arum-Paris continuum of mycorrhizal symbioses. New Phytol 163:187-200.

DIMITRI, M. J. 1972. La region de los Bosques Andino-Patagonicos. Sinopsis General. Tomo 10. INTA, Buenos Aires.

DUCKETT, J. G. and R. LIGRONE. 1992. A light and electron microscope study of the fungal endophytes in the sporophyte and gametophyte of Lycopodium cernuum with observations on the gamethophyte-sporophyte junction. Can. J. Bot. 70:58-72.

FREEBERG, J. A. 1962. Lycopodium prothalli and their endophytic fungi as studies in vitro. Am. J. Bot. 49:530-535.

GEMMA, J. N., R. E. KOSKE and T. FLYNN. 1992. Mycorrhizae in Hawaiian Pteridophytes: Occurrence and Evolutionary Significance. Am. J. Bot. 79:843-852.

GIFFORD, E. M. and A. S. FOSTER. 1989. Morphology and Evolution of Vascular Plants, 3rd Ed. WH Freeman and Company, New York.

HARLEY, J. L. and E. L. HARLEY. 1987. A Check-List of Mycorrhiza in the British Flora. New Phytol. 105:1-102.

HARLEY, J. L. and S. E. SMITH. 1983. Mycorrhizal symbioses. Academic Press, London.

HASS, H., N. T. TAYLOR and W. REMY. 1994. Fungi from the lower Devonian Rhynie Chert: mycoparasitism. Am. J. Bot. 81:29-37.

HEPDEN, P. M. 1960. Studies in vesicular-arbuscular endophytes II. Endophytes with in the pteridophyta, with special reference to leptosporangiate ferns. B Mycol. Soc. 43:559-570.

IQBAL, S. H., M. YOUSAF and M. YOUNUS. 1981. A field survey of mycorrhizal associations ill ferns of Pakistan. New Phytol. 87:69-79.

JUMPPONEN, A. 2001. Dark septate endophytes--are they mycorrhizal? Mycorrhiza 11:207-211.

JUMPPONEN, A. and J. M. TRAPPE. 1998. Dark septate endophytes: a review of facultative hiotrophic root-colonizing fungi. New Phytol. 140:295-310.

KENRICK, P. and P. R. CRANE. 1997. The origin and early diversification of land plants: a cladistic study. Smithsonian Press, Washington, D.C., USA.

KOSKE, R. E., C. F. FRIESE, P. D. OLEXIA and R. L. HAUKE. 1985. Vesicular arbuscular mycorrhizas in Equisetum. Trans. Br. Mycol. Soc. 85:350-353.

KRANZ, H. D. and V. A. R. HUSS. 1996. Molecular evolution of pteridophytes and their relationship to seed plants: evidence from complete 18S rna gene sequences. Plant Syst. Evol. 202:1-11.

MALLOCH, D. W. 1987. The evolution of mycorrhizae. Can. J. Plant Pathol. 9:398-402.

McGONIGGLE, T. P., M. H. MILLER, D. G. EVANS, G. L. FAIRCHILD and J. A. SWAN. 1990. A new method which gives and objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol. 115:495-501.

MUTHUKUMAR, T. and K. UDAIYAN. 2000. Vesicular arbuscular mycorrhizae in pteridophytes of western ghats, southern India. Phytomorphology 50:132-142.

PETERSON, R. L., H. B. MASSICOTE and L. H. MELVILLE. 2004. Mycorrhizas: Anatomy and Cell Biology. NRC Research Press, Ottawa.

PHILLIPS, J. M. and D. S. HAYMAN. 1970. Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55:158-160.

PIROZYNSKI, K. A. and D. W. MALLOCH. 1975. The origin of land plants: a matter of mycotrophism. Biosystems 6:153-164.

PRYER. M. K., E. SCHUETTPELZ, P. G. WOLF, H. SCHNEIDER, A. R. SMITH and R. GRANFILL. 2004. Phylogeny and evolution of ferns (monilophytes) with a focus on the early leptosporangiate divergences. Am. J. Bot. 91:1582-1598.

PRYER, M. K., H. SCHNEIDER, A. R. SMITH, R. GRANFILL, P. C. WOLF, J. S. HUNT and S. D. SIPES. 2001. Horsetails and ferns are a monophyletic group and the closest living relatives to seed plants. Nature 409:618-622.

READ, D. J. 1999. The ecophysiology of mycorrhizal symbiosis with special reference to impacts upon plant fitness. Pp. 133-152, In: Press, M. C., J. D. Scholes and M. G. Barker, eds. Physiological Plant Ecology. Blackwell Science, Great Britain.

READ, D. J., J. G. DUCKETT, R. FRANCIS, R. LIGRONE and A. RUSSELL. 2000. Symbiotic fungal associations in 'lower' land plants. Phil. Trans. R Soc. Lond. B 355:815-831.

READ, D. J. 1991. Mycorrhizas in ecosystems. Experientia 47:376-391.

REMY, W. T., N. TAYLOR, H. HASS and H. KERP. 1994. Four hundred-million-year-old vesicular arbuscular mycorrhiza. Proc. Nat. Acad. Sci. 9:11841-11843.

ROIG, F. A. 1998. La vegetacion de la Patagonia. Pp. 48-166, In: Correa, M. N. (Dir.)., ed. Flora Patagonica. Parte 1, Coleccion Cientifica INTA, Buenos Aires, Argentina.

SCHMID, E. and F. OBERWINKLER. 1993. Mycorrhiza-like interaction between the achlorophyllous gametophyte of Lycopodium clavatum L. and its fungal endophyte studied by light and electron microscopy. New Phytol. 124:69-81.

SCHMIDT, E. and F. OBERWINKLER. 1994. Light- and electron microscopy of the host-fungus interaction in the achlorophyllous gametophyte of Botryehium lunaria. Can. J. Bot. 72:182-188.

SITTE, P., E. W. WEILER, J. W. KADEREIT, A. BRESINSKY and C. KORNER. 2004. Strasburger, Tratado de Botdnica, 35th Ed. Ediciones Omega, Barcelona.

SMITH. S. E. and D. J. READ. 1997. Mycorrhizal simbiosis, 2nd Ed. Academic Press, San Diego.

SMITH, A. R., K. M. PRYER E. SCHUETTPELZ, P. KORALL and H. G. SCHNEIDER. 2006. The classification for extant ferns. Taxon 55:705-731.

SMITH, F. A. and S. E. SMITH. 1997. Tansley Review N 96: Structural diversity in (vesicular)arbuscular mycorrhizal symbioses. New Phytol. 137:373-388.

SMITH, S. E. and D. J. READ. 1997. Mycorrhizal symbiosis. 2nd Ed. Academic Press, London.

STEWART, W. N. and G. W. ROTHWELL. 1993. Paleobotany and the evolution of plants, 2nd Ed. Cambridge University Press, Cambridge, UK.

SWATZELL, L. J., M. J. POWELL and J. Z. Kiss. 1996. The relationship of endophytic fungi to the gamethophyte of the fern Schizaea pusilla. Int. J. Plant Sci. 157:53-62.

TREU, R., G. A. LAURSEN, S. L. STEPHENSON, J. C. LANDOLT and R. DENSMORE. 1996. Mycorrhizae from Denali National Park and Preserve, Alaska. Mycorrhiza 6:21-29.

TURNAU, K., T. ANIELSKA and A. JURKIEWICZ. 2005. Mycothallic/mycorrhizal symbiosis of chlorophyllous gametophytes and sporophytes of a fern, Pellaea viridis (Forsk.) Prantl (Pellaeaceae, Pteridales). Mycorrhiza 15:121-128.

VAN dER HEIJDEN, M. G. A., J. N. KLIRONOMOS, M. URSIC, P. MOUTOGLIS, R. STREITWOLF-ENGEL, T. BOLLER, A. WIEMKEN and I. R. SANDERS. 1998. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396:69-72.

WINTHER, J. L. and W. E. FRIEDMAN. 2007. Arbuscular mycorrhizal associations in Lycopodiaceae. New Phytol. 177:790-801.

ZHANG, Y., L. Guo and R. Liu. 2004. Arbuscular mycorrhizal fungi associated with common pteridophytes in Dujiangyan, southwest China. Mycorrhiza 14:25-30.

ZHAO, Z. 2000. The arbuscular mycorrhizas of pteridophytes in Yunnan, southwest China: evolutionary interpretations. Mycorrhiza 10:145-149.

NATALIA FERNANDEZ (1)

Universidad Nacional del Comahue, Departamento de Biologia General, Laboratorio de Microbiologia Aplicada y Biotecnologia, Quintral 1250, S. C. de Bariloche (CP: 8400), Argentina--CONICET--INIBIOMA

MARIA INES MESSUTI

Universidad Nacional del Comahue, Departamento de Botanica, Quintral 1250, S. C. de Bariloche (CP: 8400), Argentina--CONICET--INIBIOMA

SONIA FONTENLA

Universidad Nacional del Comahue, Departamento de Biologia General, Laboratorio de Microbiologia Aplicada y Biotecnologia, Quintral 1250, S. C. de Bariloche (CP: 8400), Argentina--INIBIOMA

(1) Corresponding author. Email: natifernan([e-mail]

TABLE 1. AM colonization in L. paniculatum and E. bogotense.
References: No: sample number, AM (%): mean and standard deviation
(X [+ or -] SD) of the percentage of root length colonized by AM
fungi; * positive but non-quantified sample.

                 Place of
Habitat          collection       No         AM (%)         Arbuscules

Lycopodium paniculatum
Forest           Soil              1   50.0 [+ or -] 27.7       +
                                   2           0                -
                                   3           0                -
                 Soil (edge of     4   26.2 [+ or -] 15.8       +
                   road, stony)    5           0                -
                                   6           0                -
                                   7           0                -
                 Trunk             8           0                -
Waterlogged      Soil              9   22.5 [+ or -] 18.6       +
  peat bog                        10          2.02              -
                                  11    9.3 [+ or -] 8.7        +
                                  12           0                -
                                  13   22.9 [+ or -] 9.25       -
                 Trunk            14           0                -

Equisetum bogotense
Forest           Soil (edge of     1   22.5 [+ or -] 5.7        +
                   road, stony)    2   12.9 [+ or -] 6.1        +
                                   3   10.2 [+ or -] 6.1        +
                                   4   11.5 [+ or -] 5.6        +
                                   5           +*               -
                                   6    7.6 [+ or -] 4.5        -
                                   7   12.5 [+ or -] 7.7        +
                                   8   17.1 [+ or -] 3.5        +
                 Soil (edge of     9           0                -
                   road, sandy)   10           0                -
                 Soil (coast of   11           0                -
                   the lake)      12           0                -