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Anatomy and Morphology |
2School of Biological Sciences, Queen Mary University of London, Mile End Road, London E3 4NS, UK; 3Dipartimento di Scienze ambientali, Seconda Università di Napoli, via A. Vivaldi 43, 81100 Caserta, Italy
Received for publication October 31, 2005. Accepted for publication March 20, 2006.
ABSTRACT
Thallus anatomy in three species of the primitive liverwort genus Treubia (Metzgeriidae, Treubiales) was studied by light and electron microscopy. The thallus exudes copious mucilage, a feature shared elsewhere in liverworts only with the mycotrophic subterranean axes of the allied genus Haplomitrium. The central strand in the thallus midrib has a unique histological organization and harbors an intra- and intercellular infection by a glomeromycotean fungus that is far more highly differentiated than most of the glomeromycotean associations described to date. The fungus enters the thallus via clefts in the ventral epidermis along the midrib and colonizes the parenchyma above, forming intracellular coils and prominent, relatively short-lived, hyphal swellings. Above the zone with intracellular colonization is a tissue area containing mucilage-filled intercellular spaces; here the fungus is entirely intercellular and forms abundant pseudoparenchymatous structures and, in more mature parts of the thalli, large hyphae with thick multistratose walls. Mucilage in Treubia differs in histochemistry and origin from that produced by apical papillae, via hypertrophied Golgi, in all other bryophytes. Remarkable parallels between fungal associations in Treubia, Haplomitrium, and Lycopodium, all members of very ancient lineages, suggest that these associations epitomize very early stages in the evolution of glomeromycotean symbioses.
Key Words: arbuscular mycorrhiza • Bryophyta • Glomeromycota • Metzgeriidae • mucilage secretion • phylogeny • symbiosis • Treubia
Bryophytes are widely recognized as the oldest living land plants (Shaw and Renzaglia, 2004
; Renzaglia et al., in press). Amongst the major groups of bryophytes, the Jungermanniopsida stands out as containing the widest range of morphologies (Crandall-Stotler and Stotler, 2000
; Shaw and Renzaglia, 2004
); in the plant world these are perhaps the closest living equivalent to the curious assemblage of invertebrate fossils described from the Burgess Shales (Gould, 1991
).
Most remarkable of all amongst the orders in the subclass Metzgeriidae (simple thalloid liverworts and Haplomitriales) is the Treubiales, comprising seven or eight species in the genus Treubia and two in the closely allied genus Apotreubia (Schuster and Scott, 1969
; Renzaglia, 1982
; Pfeiffer et al., 2002
; Stech et al., 2002
). Whereas a suite of morphological and developmental criteria places this order firmly in the Metzgeriidae, the restriction of oil bodies to scattered idioblasts and the unique growth form intermediate between leafy and thalloid suggest affinities with the Marchantiopsida and Jungermanniidae, respectively. Spermatid ultrastructure underlines the isolated position of the order and also indicates affinities with the Haplomitriales (Duckett and Carothers, 1983
; Carothers and Rushing, 1990
), as does the copious secretion of mucilage from the ventral surface of the thallus in Treubia and by subterranean axes in Haplomitrium (Carafa et al., 2003b
). Another pointer to the extreme antiquity of the Treubiales is their predominantly southern hemisphere distribution, interpreted as originally Pangean (Stech et al., 2000
). Placental ultrastructure (several layers of transfer cells in both generations) is yet a further indicator of a primordial position in liverworts (Carafa et al., 2003a
).
It is therefore hardly surprising that molecular data not only affirm the isolated systematic position of the Treubiales, leading to the proposition of the new clade Treubiopsida (Stech et al., 2000
), but also resolve Treubia and Haplomitrium as a sister group to all other hepatic lineages (Forrest and Crandall-Stotler, 2004
, 2005
; Heinrichs et al., 2005
).
Aside from morphological, cytological, and molecular data, all pointing to a basal position for Treubia in hepatic evolution, the unique axial anatomy of the thallus itself has yet to receive critical scrutiny. Whereas in other families, orders, and suborders in the Metzgeriidae the thallus contains either a water-conducting strand (Pallaviciniaceae, Hymenophytaceae) or a well-defined ventral fungal zone (Fossombroniales, Makinoaceae, Aneuraceae), or also lacks both these features (Blasiales, Phyllothalliaceae, Metzgeriaceae) (Read et al., 2000
), the thallus midrib of Treubia contains at least five histologically distinct zones. Following Schuster and Scott's description (1969), these comprise (1) a ventral epidermis of thick-walled cells whence arise the rhizoids, (2) 3–6 layers of small cells lacking fungi (but see Results), (3) a central strand with two ill-defined regions, (i.e., a lower region of 11–14 cell layers with abundant intercellular hyphae and an upper layer of schizolytically separated cells containing fewer hyphae), which are sometimes separated by a narrow band of nonmycorrhizal cells, (4) 6–10 layers of large, elongate pellucid cells, and (5) a cortical parenchyma of small cells.
To glean further insights into the unique organization of the Treubia thallus one must consult the original works by Goebel (1891)
, Grün (1914)
, and Stahl (1949)
because Treubia anatomy receives but scant mention and is sometimes misreported by subsequent authors. These early studies, both models of meticulous observations and well-guarded inferences, raise most intriguing questions about the fungal endophyte(s) in Treubia.
Goebel (1891)
stated that the fungal infections start from the mucilage outside the ventral surface of the thallus. He reported two kinds of hyphae within the thallus, broader ones between the cells (corresponding to zone 3a of Schuster and Scott, 1969
) and, below the intercellular fungi, a mass of cells containing a mixture of finer hyphae and "lumps" of irregular shape. The latter clearly corresponds to Schuster and Scott's zone 2, but these authors make no mention of intracellular fungal structures. Goebel described a mass of yellow intercellular material (zone 3b) above the intercellular zone, which he considered to be a product of the fungus. Goebel was unable to resolve the nature of the lumps but, in establishing their connection with the fine hyphae, assumed them to be a product of fungal secretion. He could not prove that the inter- and intracellular hyphae belonged to the same fungus, but noted that in the prothallus of Lycopodium inundatum the initially intercellular fungus subsequently grows into the cells, a feature also found in the protocorm of L. cernuum (Duckett and Ligrone, 1992
).
Grün's (1914)
and Stahl's (1949)
works confirmed and added to Goebel's observations. From the mucilage-filled ventral groove, penetrating hyphae follow the course of the cell walls before becoming intracellular. The intercellular hyphae are thick-walled with bulbous swellings, and unlike other fungus-containing hepatics (Read et al., 2000
), the rhizoids of Treubia are fungus-free. Like Goebel (1891)
, Grün and Stahl were unable to demonstrate any connection between the inter- and intracellular hyphae. Disappearance of starch from infected cells was also noted.
These descriptions underline the uniqueness of the anatomy of Treubia and suggest that the structural differentiation of its fungal endophyte(s) is without parallel in liverworts. The occurrence of a wide range of fungi in liverworts currently holds center stage in our consideration of the evolutionary origin of mycorrhizas (Nebel et al., 2004
; Kottke and Nebel, 2005
). In this context, the fungal association in Treubia is of special interest because of the basal position assigned to this taxon in plant phylogeny (Lewis et al., 1997
; Shaw and Renzaglia, 2004
; Crandall-Stotler et al., 2005
; Forrest and Crandall–Stotler, 2004
, 2005
; Heinrichs et al., 2005
).
The present paper reports a light- and electron-microscopy analysis of the fungal association in three species of Treubia, with special attention to the following hitherto unresolved issues: (a) the nature of the fungus/fungi involved, (b) the modalities of fungal penetration and spread in the thallus, (c) the origins of the intercellular material in the central strand and of the mucilage secreted from the thallus, and (d) the morphology and development of the intracellular fungal structures.
MATERIALS AND METHODS
Thalli of three species of Treubia were collected from a variety of sites in the South Island of New Zealand and identified from the characters given by Pfeiffer et al. (2002)
. Treubia pygmaea R.M. Schust. was growing over thick cushions of other bryophytes and often associated with Verdoornia R. M. Schuster in the primary forest at Rahu Saddle, west of Springs Junction at Cannibal Creek near the Lewis Pass and along the Bridle Veil Falls track at Arthur's Pass. Treubia lacunosoides Pfeiffer, Frey & Stech occurred directly on the soil, forming steep banks along tracks associated with old mine workings at Kelly Creek near Otira, and at Ross. Treubia lacunosa (Colenso) Prosk. was found on soil in very deep shade beneath tree ferns on the wet floor of the primary Dacrycarpus dacrydioides (A. Rich) de Laub (Kahikatea) forest north of Whataroa. Differences in gross morphology were not reflected in the inner anatomy of the thalli, which was essentially the same in all three species.
The specimens were transferred immediately to the laboratory and processed for light and electron microscopy. Whole plants were photographed with a Leica digital camera D-Lux (Leica, Solms, Germany) both in situ and in the laboratory. Handcut transverse sections were stained with mercury-bromophenol blue (BB) for protein or via the periodic acid-Schiff (PAS) procedure for insoluble carbohydrates (Krishnamurthy, 1999
). Controls were deamination or 4-h pre-digestion in 1% protease (Sigma, St. Louis, Missouri, USA) for protein detection and omission of the oxidation step with periodic acid for carbohydrate detection.
Preparation of samples for electron microscopy followed Carafa et al. (2003b)
. Healthy thalli were placed in the primary fixative (0.04 M piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, pH 7.0, containing 2% glutaraldehyde, 1% freshly prepared formaldehyde, and 0.75% tannic acid) and the marginal lobes, lobules, and most of the ventral mucilage removed from the central axes with a razor blade. The central axes were then cut into 1-mm-thick slices and fixed for 2 h at room temperature. Following several rinses in buffer, the samples were postfixed in 1% OsO4 in 0.08 M Na cacodylate buffer, pH 6.5, overnight at 4°C, dehydrated in an ethanol series, and embedded in Spurr's resin via propylene oxide. Thin sections were cut with a diamond knife, stained with 3% uranyl acetate in 50% methanol for 15 min and in Reynolds's lead citrate for 10 min, and observed with a Jeol (Tokyo, Japan) 1200 EX2 electron microscope operating at 20 kV.
The periodic acid–thiocarbohydrazide–silver proteinate (PATAg) test for carbohydrates was performed on sections collected on 300-mesh gold grids following the protocol of Roland and Sandoz (1969)
.
For scanning electron microscopy, slices of the central strand were taken through a 1:1 ethanol : acetone series to remove cytoplasm, osmicated for 48 h in aqueous 2% OsO4, dehydrated in anhydrous ethanol, critical-point dried, and viewed in a Hitachi (Tokyo, Japan) S570 scanning electron microscope.
For light microscopy, 0.5-µm-thick sections were cut with a diamond histoknife, stained with 0.5% toluidine blue, and photographed using bright-field or differential interference contrast optics with a Zeiss (Jena, Germany) Axioskop light microscope , fitted with a Sensicam QE digital photocamera (Applied Scientific Instrumentations, Eugene, Oregon, USA).
RESULTS
General morphology and light microscopy
The gross appearance of the thalli of Treubia, with their marginal lobes and smaller dorsal lobules, is illustrated in Fig. 1a, c. When the thalli are removed from their native substrata, a copious mucilage exuding from the ventral surface of the midrib region becomes visible (Fig. 1b). Mucilage is particularly abundant in the younger part of the thalli, but production declines in the older parts (Fig. 1d). Indeed the scale of production is most remarkable; within 30 min of placing freshly collected thalli in water, the volume of the mucilage virtually doubles (Fig. 1d, e). If this mucilage is removed, it is replenished within 1 h. The ventral half of the thallus midrib is occupied by a prominent central strand (Fig. 1e). The inner anatomy of this strand is almost the same in all three species examined here, except for the number of cells forming the various regions, and corresponds very closely to descriptions by previous authors (Goebel, 1891
; Grün, 1914
; Schuster and Scott, 1969
).
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) have very small or no intercellular spaces. The cells forming the lowest 2–3 layers in this area are fungus-free (Fig. 2c), whilst those above contain abundant intracellular fungal structures with the exception of the oil-body cells, which are not infected (Fig. 2c, d). The first infected cells, detected in the nerve region 4–8 mm behind the thallus apex in T. lacunosoides, contain hyphae of uniform diameter (Fig. 3a). Moving slightly further from the thallus apex, densely staining fungal swellings up to 15 µm in diameter are found along with thin hyphae (Fig. 3b). In more mature parts of the thallus, these structures rapidly become more frequent, and at a certain distance from the apex (about 15–20 mm in T. lacunosoides), virtually all the cells of the intracellular fungal zone contain several profiles of fungal swellings, most of which are collapsed (see the electron microscopy section), whilst the normal hyphae in these cells are still healthy (Figs. 2d, 3c). Moving to older parts of the thallus, an increasing number of cells contain totally collapsed fungal remains.
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). Near the thallus apices the intercellular spaces are packed with thin-walled hyphae (Fig. 3d), but progressing rearward (10–20 mm from apex in T. lacunosoides) these give way to thick-walled fungal structures (Fig. 3e). Two to three layers of closely packed cells separate this extracellular fungal region from a more dorsal region of 6–12 cell layers with prominent intercellular spaces filled with mucilage but lacking fungal hyphae (zone 3b of Schuster and Scott, 1969
) (Fig. 3f). Above the central strand are 3–4 layers of vacuolated cells that are slightly elongated longitudinally and lacking intercellular spaces (stratum 4 of Schuster and Scott, 1969
) (Fig. 2a, b). The dorsal parenchyma (stratum 5 of Schuster and Scott, 1969
) comprises smaller isodiametric and highly vacuolated cells. PAS gave intense purple staining of the hepatic cell walls, the intercellular spaces, and the external mucilage investitive, whereas BB lightly stained the external mucilage and more intensely the intercellular spaces in the region lacking fungus. Following treatment with protease, BB staining was completely negative.
Electron microscopy
The ventral epidermal cells are highly vacuolate and have strongly thickened outer walls (Fig. 4a). Irregular splitting along the middle lamella between these cells opens into mucilage-filled clefts that expand into the parenchyma tissue immediately above (Fig. 4a). The mucilage accumulated in the intercellular spaces of the internal parenchyma exudes from the thallus through these clefts to completely invest the thin-walled rhizoids and epidermal surface (Fig. 4a, d); minor amounts of mucilage are also seen exuding through breaks in the thin osmiophilic boundary layer outlining the outer walls of epidermal cells (Fig. 4c). The dorsal epidermal cells of the midrib region and both epidermes of the wings are highly vacuolate, and they bear no evidence of producing mucilage. Fungal hyphae, abundant in the external mucilage, penetrate the thallus parenchyma via the clefts among ventral epidermal cells (Fig. 4a, b), whilst the rhizoids remain completely fungus-free, at least while alive. In the cell layers immediately above the epidermis, the fungus remains entirely extracellular (Fig. 4b) and may proliferate in mucilage-filled intercellular spaces that communicate with the exterior (Fig. 4e). From this area the hyphae enter the more internal parenchyma cells (Fig. 4f). The original hepatic walls are broken down at the entry points, but inside the liverwort cells, additional host wall material is deposited around the hyphae.
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The intercellular spaces in the middle region of the central strand in young parts of the thallus of Treubia are packed with a network of highly branched, thin-walled hyphae with an almost pseudoparenchymatous growth pattern (Fig. 8a). Some are packed with glycogen rosettes (Fig. 8a), others (probably older) contain abundant lipid deposits (Fig. 8b). Also present are endosymbiotic bacteria (not illustrated) like those in intracellular hyphae (Fig. 6b). Moving rearward along the thallus, the intercellular spaces have increasing numbers of collapsed hyphae together with hyphae with thicker walls, up to 6 µm in diameter (Fig. 8c). Further from the apex (about 8–10 mm in T. lacunosoides), the thick-walled hyphae become packed with lipid deposits and expand to form cylindrical structures 15–20 µm in length (Fig. 8d). In mature parts of the thallus, the intercellular spaces are almost completely filled with fungal structures packed with lipid and with electron-opaque, multistratose cell walls (Fig. 8e). Together, the walls comprise up to 10 continuous inner layers, each less than 1-µm thick, surrounded by two or three discontinuous strata each approximately 0.5-µm thick. Also present in these spaces are a few healthy thin-walled hyphae growing amidst others that are either empty or collapsed. New hyphae growing out from thick-walled structures were observed occasionally (Fig. 8f).
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DISCUSSION
The central strand and fungal association in Treubia are unique
The entire thallus of Treubia, apart from the slightly elongated cells above the central strand, is made up of thin-walled isodiametric cells. The complex differentiation of the thallus in this liverwort genus, however, depends on the presence of mucilage-filled intercellular spaces and the unique distribution of the fungal endophyte in the central strand. The present study demonstrates that a fungal association is regularly present in all three species examined and is probably a constant feature of the biology of Treubia in nature. Demonstration of hyphal continuity between the intra- and extracellular components of the association, together with a suite of ultrastructural commonalities, confirms the ideas of Goebel (1891)
and Grün (1914)
that these are one and the same fungus. In this study, it was also noted that the intracellular component was extremely sensitive to disturbance and degenerated completely within a few days after collection, even if the samples were kept in a cool room at 8°C. This might explain inconsistencies between previous reports.
The fungal endophyte in Treubia has clear affinities with glomeromycotean associates in higher plant arbuscular mycorrhizas (Smith and Read, 1997
), as well as in other liverworts (Ligrone and Lopes, 1989
; Read et al., 2000
; Russell and Bulman, 2005
), notably Haplomitrium (Carafa et al., 2003a
), in hornworts (Ligrone, 1988
; Schüßler, 2000) and in pteridophytes (Duckett and Ligrone, 2005
; Schmid and Oberwinkler, 1993
, 1994
, 1995
).
As typical of the aforementioned associations, the cytological responses, absence of damage to the host cells, and the restriction of the fungus to specific zones within the thallus of Treubia indicate a high level of compatibility between the partners. The initial stages in intracellular fungal colonization, involving overgrowths of host wall material around hyphal entry sites and proliferation of hyphae surrounded by an interfacial matrix and perifungal membrane both of host origin, also follow the usual pattern of glomeromycotean infections. The fungal association in Treubia, however, with intra- and intercellular infection of distinct and specific tissue areas, is far more highly differentiated than any of the glomeromycotean associations with photosynthetic plants described to date and is comparable in complexity to highly differentiated associations in achlorophyllous angiosperms (Imhof, 1999
, 2003
).
As in Haplomitrium (Carafa et al., 2003b
) but unlike marchantialean liverworts (Ligrone and Lopes, 1989
; Russell and Bulman, 2005
), there is no evidence in Treubia of longitudinal spreading of the fungus along the thallus nerve nor of repeated cycles of intracellular infection. A further similarity with Haplomitrium is that initial stages of fungal development are found only in the apical region of the thallus, whilst more mature parts contain only advanced stages of fungal colonization. This indicates that only the youngest part of the thallus is receptive to fungal infection.
Intracellular fungal growth in Treubia does not produce typical arbuscular systems but rather a mass of coiled hyphae with short lateral branches. Development of intracellular coils but no arbuscules has been reported for glomeromycotean associations in achlorophyllous gametophytes (and rhizomes) of Psilotum and Tmesipteris (Peterson et al., 1981
; Duckett and Ligrone, 2005
), Lycopodium clavatum (Schmid and Oberwinkler, 1993
), and Botrichium lunaria (Schmid and Oberwinkler, 1994
) as well as in the roots of some parasitic angiosperms (Imhof, 1999
, 2003
). In the photosynthetic gametophytes of Lycopodium cernuum (Duckett and Ligrone, 1992
) the fungus forms moderately branched hyphal coils similar to those in Treubia, whilst more typical arbuscules have been reported in the gametophytes of the Gleicheniaceae (Schmid and Oberwinkler, 1995
). Intracellular fungal development in Treubia proceeds hand-in-hand with proliferation of the host cytoplasm, that contains prominent Golgi bodies, whilst the starch content in plastids, abundant in uninfected cells, is reduced. In contrast to other glomeromycotean associations, in which infected cells lack starch, infected cells in Treubia often retain small amounts of starch. Bacterial endosymbionts are a further ubiquitous feature of glomeromycotean fungi, and molecular studies are now required to find out whether these belong to the genus Burkholderia as seen in Gigaspora in seed plants (Bianciotto et al., 2000
).
The most remarkable feature of the intracellular infection in Treubia are the swellings developing at the end of hyphal branches. These almost certainly correspond to the structures previously described by Goebel (1891)
, Grün (1914)
and Stahl (1949)
, the present study confirming their fungal origin, suspected but not proven by these authors. The fungal swellings in Treubia are identical in every respect to those described previously in Haplomitrium and referred to as "fungal lumps" (Carafa et al., 2003b
). In contrast to the so-called vesicles, long-lived structures with thick walls and abundant lipid, of common occurrence in other associations with glomeromycotean fungi (cf. Bonfante-Fasolo, 1984
; Ligrone, 1988
; Schmid and Oberwinkler, 1993
; Imhof, 1999
, 2003
) and probably functioning as perennating organs (Bonfante-Fasolo, 1984
; Harrison, 1997
), the fungal lumps in Haplomitrium and Treubia go through a developmental cycle that concludes with cytoplasmic degeneration and collapse. The observation that initial stages of lump development are only present in the youngest part of the thallus, whilst cells in the area just behind this contain only collapsed lumps alongside healthy coil hyphae, indicates that lump development is a relatively fast process restricted to an early phase of intracellular colonization. The ghosts of crystals visible in advanced stages of degeneration are most likely calcium oxalate dissolved during fixation. The intercellular infection in Treubia has no counterpart elsewhere in hepatics, where the fungi are exclusively intracellular, and differs sharply from the fungal infection in Phaeoceros, where isolated hyphae colonizing intercellular spaces in the thallus parenchyma have been observed (Ligrone, 1988
). Indeed, the glycogen-packed pseudoparenchymatous hyphae in Treubia superficially resemble ectomycorrhizas, though here the fungi are basidiomycetes and are restricted to the outermost cortical parenchyma (Smith and Read, 1997
). The only other examples of glomeromycotean endophytes forming pseudoparenchymatous hyphal masses in intercellular spaces are recorded to date in the sporophyte protocorm and root nodules in Lycopodium cernuum, where the spaces are schizogenous (Duckett and Ligrone, 1992
) and in the storage tissue in the gametophyte of L. clavatum, where the spaces may be lysigenous in origin (Schmid and Oberwinkler, 1993
). These comprise glycogen-packed hyphae and thick-walled vesicles lacking the multilayered structure observed in mature intercellular fungal swellings in Treubia.
The development of intercellular fungal swellings initially follows the same course as seen for the intracellular swellings, but subsequently these structures develop thickened multistratose cell walls and accumulate abundant lipid reserves, remaining alive even after the death of most intercellular hyphae in the more mature parts of the thallus. Fungal structures with similar multistratose walls have been noted previously in the external mucilage around the subterranean axes in Haplomitrium (Carafa et al., 2003b
). The observation that the intercellular fungal swellings in the thallus of Treubia are able to germinate producing thin-walled hyphae suggests that they are long-lived perennating structures functionally equivalent to the vesicles produced in other glomeromycotean associations (Bonfante-Fasolo, 1984
; Harrison, 1997
). They probably survive the death and decay of the old parts of the thallus and remain in the soil as inoculum awaiting new host plants.
In marked contrast to the highly differentiated central strand, the cells above the upper mucilage zone are highly vacuolated unremarkable parenchyma. Given the dimensions of Treubia thalli, the absence of cells with "food-conducting cytology," viz., containing vesicles and mitochondria aligned along endoplasmic microtubules, as seen in many other thalloid liverworts (e.g., vAsterella, Marchantia, Conocephalum, Monoclea) and in Haplomitrium (Ligrone and Duckett, 1994
; Ligrone et al., 2000
), is somewhat surprising.
Functional and evolutionary aspects
The reduction of starch in infected cells, also observed in other glomeromycotean associations with photosynthetic hosts (Bonfante-Fasolo, 1984
; Ligrone, 1988
; Ligrone and Lopes, 1989
), is consistent with the notion of a transfer of carbohydrates from the plant to the fungus (Smith and Read, 1997
). The large amounts of lumps produced in the intracellular fungal zone certainly account for a significant fraction of the intracellular fungal biomass in the thallus of Treubia. It is likely, therefore, that these structures play a significant role in host–fungus physiological relationships. As suggested in Haplomitrium (Carafa et al., 2003b
), the massive proliferation and subsequent lysis of intracellular lumps might be a source of nutrients of fungal origin (notably nitrogen) to the host plant. It must be noted, however, that the possibility of transfer of organic carbon from fungus to plant in glomeromycotean associations with photosynthetic hosts has recently been challenged (Pfeffer et al., 2004
).
Perhaps the most equivocal aspect of this study concerns the nature, origin and possible function of the copious mucilage exuding from the thallus in Treubia and, by analogy, from the subterranean axes in Haplomitrium (Carafa et al., 2003b
). This kind of mucilage differs in both the scale of its production and mode of origin from that secreted by apical mucilage hairs or papillae in mosses and liverworts, including also Haplomitrium and Treubia (Bonnot and Hebant,1970
; Galatis and Apostolakos, 1977
; Ligrone, 1986
; Duckett et al., 1990
; R. Ligrone and J. G. Duckett, unpublished data). Mucilage secretion in these glandular structures involves Golgi-derived PATAg-positive vesicles that discharge their contents by exocytosis and contain no appreciable amounts of protein. The mucilage exuding from the thallus in Treubia appears to be produced mostly by the cells facing the intercellular spaces in the central strand. These cells contain abundant smooth endoplasmic reticulum but few inconspicuous dictyosomes, thereby suggesting that mucilage secretion here follows a different pathway. The strongly positive reaction to PAS of the ventral mucilage in Treubia demonstrates the presence of insoluble carbohydrates, whilst staining with BB is indicative of the presence of a protein component. In line with cytological differences, the negative reaction to PATAg staining points to a different composition from the product of mucilage hairs or papillae (Bonnot and Hébant, 1970
; Ligrone, 1986
; J. G. Duckett and R. Ligrone, unpublished data), although this method has too low specificity (Krishnamurthy, 1999
) to be used for positive identification of polysaccharidic components. Noteworthy, the mucilage investing the subterranean axes in Haplomitrium was strongly positive to PATAg staining (Carafa et al., 2003b
). The lack of reaction to a monoclonal antibody against xylan (R. Ligrone et al., unpublished data) suggests that the mucilage in Treubia does not contain the hydrophilic xylan-cellulosic complexes known in higher plants (Reis et al., 1994
).
Once secreted, the mucilage is able to swell to considerable larger volumes by absorbing external water. It may therefore function as a water-storing system protecting the thalli from occasional drying conditions. The observation that mechanical disturbance of the thalli stimulates mucilage production also suggests a role as a deterrent against herbivory. Another interesting feature of the Treubia and Haplomitrium mucilage is the absence of microrganisms, apart from the fungal endophytes, perhaps indicative of antimicrobial properties.
Examination of living specimens of other taxa considered to produce copious mucilage (Phyllothallia, Monoclea, Verdoornia) (Crandall-Stotler et al., 2005
) reveals that this derives exclusively from mucilage papillae and not from the general surface of the thalli. The only other places in bryophytes where mucilage is produced on a comparable scale as in Treubia and Haplomitrium are the schizolytic intercellular spaces harboring Nostoc colonies in the thallus of Blasia and of the hornworts, and the internal cavitities of the thallus in some hornworts, for example Anthoceros (Duckett et al., 1977
; Honegger, 1980
). In the former, the cytological evidence points to the mucilage as originating from the cyanobacterium because the adjacent host cells lack prominent Golgi and ER; the latter situation has never been investigated. The thickened outer walls of the ventral epidermal cells in Treubia most likely provide mechanical support for the clefts through which the fungus enters the thallus and mucilage is exuded. However, as in Haplomitrium (Carafa et al., 2003b
), mucilage is also secreted from ventral epidermal cells. The breaks in the lower epidermis of Treubia recall the openings leading to the Nostoc-containing chambers in the thalli of hornworts (Parihar, 1965
). These, however, are bordered by distinctive cells sometimes compared to stomatal guard cells, whilst the Treubia fissures are irregular and only clearly apparent in section. Unlike the ubiquitous occurrence of schizogenous intercellular spaces in vascular plants, these are far less frequent in bryophytes. Indeed the only well-documented examples are those associated with the stomata in the sporophytes of mosses and hornworts and the intercellular spaces in the inner cortex of the sporophyte foot and seta in mosses (Hebant, 1977
; Ligrone and Gambardella, 1988
). The air chambers in the thallus of the Marchantiales develop by overgrowth of subepidermal cells and not by schizolysis (Apostolakos and Galatis, 1985
). Thus the intercellular spaces in the thallus midrib of Treubia, albeit filled with mucilage, appear to be the only well-documented instance in liverworts. Similar mucilage-filled interstices have been observed in the massive setae of Treubia and a few other liverwort genera, for example, Monoclea, Wettsteinia, and Schistochila (J. G. Duckett and R. Ligrone, unpublished data).
Concluding remarks
The thallus organization and fungal association in Treubia have a degree of structural specialization unknown elsewhere in liverworts and as such provide a most striking example of plant–fungus coevolution (Brundett, 2002
). The question whether the highly distinctive pattern of fungal morphogenesis in Treubia depends on the involvement of a specific mycobiont or on control by the host requires identification of the fungus by molecular analysis and resynthesis experiments. Similarities with the fungal association in Haplomitrium might reflect sharing of the same or a similar type of mycobiont and are consistent with recent cladistic analyses indicating Treubia and Haplomitrium as a monophyletic lineage that is sister to all other liverworts (Forrest and Crandall-Stotler, 2004
, 2005
; Heinrichs et al., 2005
). Molecular, morphological, and fossil evidence all point to Devonian (Hass et al., 1994
; Read et al., 2000
; Brundett, 2002
, 2004
) or perhaps even Ordovician (Blackwell, 2000
; Redecker et al., 2000
) origins for glomeromycotean associations with vascular plants and indicates that these predated the evolution of roots. Taking account of the basal positions assigned to Haplomitrium and Treubia in both morphology- and molecular-based phylogenetic reconstructions (Forrest and Crandall-Stotler, 2004
, 2005
; Heinrichs et al., 2005
) and the basal designation of liverworts in land plant evolution (Lewis et al., 1997
; Shaw and Renzaglia, 2004
), their fungal associations could well be even more ancient than those in rootless vascular plants. The fungus-free rhizoids in Treubia and the absence of rhizoids in Haplomitrium suggest that their fungal relationships were established even before the evolution of rhizoids because in all other liverwort–fungal associations described so far these are the main, if not only, route for fungal penetration (Read et al., 2000
). The same might apply to lycophytes, the basal tracheophyte lineage (Pryer et al. 2001
) because in Lycopodium cernuum the gametophyte rhizoids and sporophyte root hairs are both fungus-free (Duckett and Ligrone, 1992
). It should be noted, however, that it is far from certain that sequencing will provide unequivocal support for the antiquity of these associations. Identification of the endophyte in Marchantia as belonging to a derived clade in the genus Glomus, forming most of the arbuscular mycorrhizas in higher plants, suggests secondary acquisition and hence a relatively recent origin for this association (Russell and Bulman, 2005
). This is perhaps not unexpected because Marchantia is an advanced genus in the Marchantiales (Forrest and Crandall-Stotler, 2004
, 2005
), colonizing predominantly ephemeral weedy habitats in contrast to the extremely ancient ecosystems where Treubia and Haplomitrium grow (Schuster and Scott, 1969
). It is also noteworthy, in the evolutionary context, that another New Zealand liverwort, Verdoornia, formerly considered an isolated and ancient monotypic genus (Schuster, 1999
), is now strongly supported in molecular trees as sister to the Aneuraceae, a crown family in the Mezgeriales (Forrest and Crandall-Stotler, 2004, 2005). Verdoornia and the Aneuraceae share basidiomycete associations that are almost certainly of recent origin (Ligrone et al., 1993
; Kottke et al., 2003
; Kottke and Nebel, 2005
; Nebel et al., 2004
; R. Ligrone and J. G. Duckett, unpublished data on Verdoornia).
FOOTNOTES
1 This work was funded in part by grants from the Seconda Università di Napoli and Regione Campania (LR 5, 2003) and was performed using facilities at the CISME (University of Naples "Federico II", Italy). J. G. Duckett was supported in New Zealand by an overseas travel grant from the Royal Society (UK) and by the New Phytologist Trust. The authors thank the Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand, for providing laboratory facilities and the New Zealand Department of Conservation for granting collecting permits. Collection of the specimens used in this study was facilitated by local knowledge of the sites by Professor B. Butterfield (University of Canterbury) and D. Glennie (Landcare, Lincoln, New Zealand). The authors also thank L. Chittka (Queen Mary, University of London) for his perceptive translation of the archaic German in the original papers by Goebel and Grün, and N. Andrews (Christchurch) and K. Pell (Queen Mary, University of London) for technical assistance. 
4 Author for correspondence (roberto.ligrone{at}unina2.it
)
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