Mycorrhizal-like interaction between gametophytes and young sporophytes of the fern Dryopteris muenchii ( Filicales ) and its fungal endophyte

The morphology of a Glomus-like fungus-host interaction in chlorophyllous gametophytes and young apogamic sporophytes of Dryopteris muenchii A.R. Sm. was studied from ferns cultivated in laboratory, using soil as substrate. An aseptate fungus colonized the gametophytes’ tissue through the rhizoids, developing vesicles. The fungus penetrated the young sporophytes primary roots by developing appressoria. It spread forming interand intra-cellular hyphae through the epidermis and the outermost cortical cell layers, where it formed vesicles, hyphal coils-like and arbuscules. The fungus hyphae never colonized the gametophytesporophyte cellular junction. The fungal structures observed on D. muenchii during this study, are rather similar to those reported for the plant host-arbuscular mycorrhizal fungus (AMF) interaction, where the AMF described belonged to Phylum Glomeromycota. Therefore, this study is a contribution to the scarce knowledgement of the association between AMF and chlorophyllous gametophytes and young apogamic sporophytes of ferns. Rev. Biol. Trop. 56 (3): 1101-1107. Epub 2008 September 30.

Arbuscular mycorrhizal fungi (AMF) are associated with ca.80-85% of living terrestrial plants (Pirozynski 1981, Dickson 2004), and are considered to be ecologically important for most vascular plants because they improve the uptake of specific nutrients by the host, other functions attributed to the fungi include the promotion of plant growth hormones production, protection of host roots from pathogens, and favors the increase of solubility of soil minerals, among other benefits (Remy et al. 1994, Newsham et al. 1995, Merryweather and Fitter 1996, 1998a, 1998b).Blackwell (2000) suggested that symbiotic relationships of AMF and the roots of higher plants were essential in moving plants to land.The fossil record shows that some Devonian fossil plants (genera Aglaophyton, Rhynia and Asteroxylon, 400 Myr) already had vesiclelike structures similar to those of Glomus (Pirozynski 1981, Remy et al. 1994), which is consistent with the hypothesis that arbuscular mycorrhizas (AM) were instrumental in the colonization of land by ancient plants (Taylor et al. 1995).
Therefore, the aim of this study is to contribute to the knowledge of the AMF-host association by describing the morphology of a Glomus-like fungus-host interaction in chlorophyllous gametophytes and young apogamic sporophytes of Dryopteris muenchii A. R. Sm. (Dryopteridaceae), a narrow endemic Mexican fern species.
Although there are studies of this type in other regions of the world, as far as we know, this is the first one done in Mexico and it is related to the knowledge of the state of D. muenchii wild populations.

Dryopteris muenchii in Mexico
Family Dryopteridaceae comprises 16 genera and ca.400 species.In Mexico, this family is represented by eight genera: Arachniodes, Didymochlaena, Dryopteris, Olfersia, Phanerophlebia, Polybotrya, Polystichum and Stigmatopteris (Moran 1995).Genus Dryopteris Adans.("shield fern") occurs in temperate and tropical regions of the world, 300-3050 m.o.s.l., with ca.225 species.Its highest diversity occurs in Eastern Asia, while only 30 species are found in the New World, 13 of them occurring in Mexico (Mickel and Smith 2004).
According to Mickel and Smith (2004), D. muenchii A.R. Sm. is endemic to Mexico; it has only been collected in the states of Hidalgo and Chiapas; this species is found in cloud forest (2000-2850 m.o.s.l).

MATERIALS AND METHODS
Dryopteris muenchii spores were collected from fertile sporophytes, using paper bags, from the Paraje Bonabil, state of Chiapas, Mexico (5 km on Matzab deviation, San Cristóbal de las Casas-Tenejapa road); voucher specimens are deposited at the Herbario Metropolitano-UAMIZ (A.Mendoza, R-182, R-244).Fern spores were sown, without sterilization, in 16 pots (5 cm in diam.), filled with 30 g of soil collected at 0-30 cm depth.Gametophyte cultures were maintained during nine months under laboratory conditions, inside transparent polyethylene bags for avoiding dehydratation and contamination, and using a 12 h light-12 h darkness photoperiod (artificial light, 75 watts), with a daily mean temperature of 25 °C.Pots were watered every ten days with distilled water.Gametophytes, with and without young sporophytes, were carefully washed with distilled water, and fixed with FAA (formol 5%, glacial acetic acid 5%, ethylic alcohol 45%, and distilled water).

RESULTS
D. muenchii gametophytes are chlorophyllous with a cordate-spatulate shape.The midrib, with 3-4 cells thick, projects numerous rhizoids (Figs. 1 and 2).This species is considered apogamic because, in vitro, it does  not form archegonia and the sporophytes developed have an apogamic origin.The antheridia are produced in the middle region or in the margins of the prothallia (Fig. 3).From 450 gametophytes examined, 51.6% of the prothallia developed apogamic sporophytes (Fig. 1), 43% were asexual (without antheridia and archegonia), and 5% developed only antheridia without sporophytes.Only 4% of the gametophytes, with or without sporophytes, were mycorrhizal.very few AMF spores were reported amongst the gametophyte rhizoids, the spores were organized in groups of 5, pairs or singles, adhering to each other by common peridial hyphae.AMF spores are globose to subglobose, varying in color from orange-to redbrown (Figs.4-6).
Gametophyte rhizoids are unicellular and hyaline with 23 µm to 50 µm in diameter.The endophytic fungus enters the host tissue through the rhizoids developing appresoria in the rhizoid wall at the penetration point.Hyphae, 2.5 to 5 µm in diameter, are found inside the rhizoids; these hyphae occur near the rhizoid apex up to the prothallic cells of the gametophyte, where they branch out.Rhizoidal hyphae are aseptate; frequently, these hyphae give rise to oval-shaped vesicles of ca. 15 x 20 µm in diameter, which are densely colored (Fig. 7).
Arbuscular mycorrhizal fungi (AMF) produced inter-and intracellular hyphae on the gametophyte midrib and wings prothallic cells; hyphae branch dispersing over a bigger surface (Figs. 8 and 9).
The meristematic region, placed in the midrib of the gametophyte, and the antheridia are regularly free of infection.The fungus never colonizes the sporophyte foot cells.The primary roots of the young sporophyte are frequently found colonized by a fungal endophyte, morphologically similar to the one described within the gametophyte.The fungus enters the host tissue through the roots developing appessoria in the rhizodermis surface at the penetration point (Fig. 10).
The fungus penetrates the primary roots and spreads forming intra-cellular linear grouped hyphae in the rhizodermis and in the outermost cortical cell layers of the roots, developing hyphal enlargements at the distal hyphal tip, increasing the AMF surface; these structures look like incipient arbuscles (Fig. 11), which colored intensively with trypanblue.Structures similar to hyphal coils were observed, as well, inside the inner root cortex (Fig. 11).
Finally, it is important to mention that the endophitic fungi associated to the young sporophytes, did not infected the gametophyte talus.
Apomictic plants are usually adapted to microhabitats, which could explain, in part, the fact that D. muenchii is a narrow endemic fern, and apomixis is also associated with polyploidy, phenomenon rather common in ferns.However, there are no studies relating both AMF and apogamic ferns, in functional and evolutionary terms.
From 450 gametophytes, only 4% of them, with or without sporophytes, were mycorrhizal.The scarce AMF spores, obtained from the pots, closely resemble to those of Glomus globiferum: the spores were organized in groups of 5, pairs or singles, adhering to each other by common peridial hyphae, having a globose to subglobose shape and varying in color from orange-to red-brown (Koske and Walker 1986).
We also found similar fungal structures than the ones found in Gleichenia bifida (Willd.)Spreng by Schmid and Oberwinkler (1995): the endophytic fungal hyphae were aseptate and spread inter-and intra-cellularly, they penetrated the host tissue through the rhizoids, developed vesicles in both the rhizoidal and the prothallic cells.
Within the gametophyte chlorophillic cells, intracellular fungal hyphae were reported; the hyphae branched covering most of the prothallic cells and, in some cases, the hyphae managed to trespass the cell wall, establishing inter-cellular hyphae; however, no arbuscles were registered.
In spite of this, the fungus penetrated the sporophyte primary roots and spread forming intra-and inter-cellular linear grouped hyphae in the rhizodermis and in the outermost cortical cell layers of the roots, developing "arbusculeshaped structures" and hyphal like-coils.The fungus continued to spread by linear or coiled hyphae; this might be considered a fungal adaptation to the limited growth of both the gametophytes and the sporophytes in comparison to the root system of the vascular plants root system.
The fungal structures observed on D. muenchii during this study, are rather similar to those reported for the plant host-AMF interaction, where the AMF described belonged to Glomeromycota.Therefore, this study is a contribution to the scarce knowledgement acquired of the association between AMF and chlorophyllous gametophytes and young apogamic sporophytes of ferns.