Introduction
The lichen symbiosis is a biologically intimate collaboration that has arisen many times independently within different clades of higher fungi (Lücking et al. Reference Lücking, Hodkinson and Leavitt2017) in combination with diverse lineages of algal partners (Sanders & Masumoto Reference Sanders and Masumoto2021). Since the relationship was first recognized (Schwendener Reference Schwendener1868, Reference Schwendener1869), substantial interest has centred on the zones of contact between fungal and algal cells. It was evident that in lichens a fungus exploits a photosynthetic organism as a food source, a situation for which many other examples were already well known (de Bary Reference de Bary1866). Based on comparisons with plant pathogenic fungi, mycobiont penetrations into algal cells were identified as haustoria. Initially, their presence was thought to be exceptional; few lichens appeared to have them (Bornet Reference Bornet1873; Paulson & Hastings Reference Paulson and Hastings1920; Nienburg Reference Nienburg1926). However, careful observations and more thorough surveys (Tschermak Reference Tschermak1941; Plessl Reference Plessl1963) eventually revealed that fungal penetration into the algal cell lumen (intracellular) was quite common in simple crustose lichens lacking organized tissues, while in more complex forms with differentiated fungal and algal layers, the mycobiont frequently bored into the algal cell wall without fully traversing it (intraparietal) or made superficial contact only (appositional). The observations and line drawings made by Tschermak (Reference Tschermak1941) and Plessl (Reference Plessl1963), near the limits of the light microscope’s resolution, were corroborated in nearly all essential details by subsequent transmission electron microscope studies (e.g. Moore & McAlear Reference Moore and McAlear1960; Chervin et al. Reference Chervin, Baker and Hohl1968; Peveling Reference Peveling1968; Galun et al. Reference Galun, Paran and Ben-Shaul1970, 1971a; Honegger Reference Honegger1984, Reference Honegger1986a; Matthews et al. Reference Matthews, Tucker and Chapman1989). However, the significance of the penetrative contacts, which so often do not even enter the algal cell lumen, has remained enigmatic.
Although ‘haustorium’ is usually defined as a specialized hyphal branch that serves to absorb food (Kirk et al. Reference Kirk, Cannon, David and Stalpers2001), in lichens the term is routinely applied to penetrative structures that are unlikely to play a central role in obtaining nutrition. Physiological studies of substance transfer in lichens suggest mass leakage of carbohydrate by the algal symbionts (Richardson et al. Reference Richardson, Hill and Smith1968; Smith et al. Reference Smith, Muscatine and Lewis1969; Smith Reference Smith, Cook, Pappas and Rudolph1980), rather than a directed removal via fungal absorptive structures. Autoradiographic studies confirm that carbohydrate flows across the entire algal wall surface, not merely through the haustorial interface (Jacobs & Ahmadjian Reference Jacobs and Ahmadjian1971; Hessler & Peveling Reference Hessler and Peveling1978). Applying quantitative arguments, Collins & Farrar (Reference Collins and Farrar1978) went so far as to conclude that lichen haustoria play no role at all in carbohydrate transfer. Further research revealed a fine layer of mycobiont-secreted hydrophobic materials that coats the exterior wall surfaces of the contacting symbionts, channelling the flow of leaked photosynthate and any other solutes apoplastically between them (Honegger Reference Honegger1986b, Reference Honegger1991; Trembley et al. Reference Trembley, Ringli and Honegger2002). This discovery provided a more lucid understanding of how substances released over the entire cell surface can be passed efficiently between the symbionts. What has remained unclear is whether the morphology of the contact zone (intracellular, intraparietal, or appositional) has any real implications for inter-symbiont transfer, or whether the diversity of those interfaces might be better explained by mechanical factors and/or phylogenetic history.
If the algal symbiont is releasing photosynthate over its entire cell surface, the fungus would seem to have little to gain nutritionally by penetrating into algal cells. On the other hand, the dense fungal and algal cell walls, although theoretically porous, might to some extent slow down the flow of solutes between symbionts (Tschermak-Woess Reference Tschermak-Woess1988). If that is the case, a reduction in wall thickness could conceivably improve transfer efficiency. Although attention is usually focused on the degree of degradation of the algal cell wall by the fungal haustorium, several authors have also noted substantial thinning of the fungal wall in zones of symbiont cell contact (Chervin et al. Reference Chervin, Baker and Hohl1968; Malachowski et al. Reference Malachowski, Baker and Hooper1980; Tschermak-Woess Reference Tschermak-Woess1983; Honegger Reference Honegger1984; Büdel & Rhiel Reference Büdel and Rhiel1987). Extreme cases have been reported in TEM studies of Verrucariaceae by Galun et al. (Reference Galun, Paran and Ben-Shaul1971b, Reference Galun, Kushnir, Behr and Ben-Shaul1973), Kushnir & Galun (Reference Kushnir and Galun1977), and Galun (Reference Galun and Galun1988), who described the complete dissolution of the fungal cell wall surrounding the haustorium, such that a naked fungal protoplast emerged into a space formerly occupied by the dissolved wall materials. However, their published micrographs are not unambiguously supportive of this interpretation. In those figures, the area identified as a ‘space’ surrounding the putatively naked protoplast is a highly electron-transparent zone that is continuous and indistinguishable from the equally electron-transparent band clearly corresponding to the fungal cell wall elsewhere in the micrographs (fig. 4 in Galun et al. (Reference Galun, Paran and Ben-Shaul1971b); figs 5–7 in Galun et al. (Reference Galun, Kushnir, Behr and Ben-Shaul1973); fig. 3 in Kushnir & Galun (Reference Kushnir and Galun1977)). Perhaps some clarifying detail was lost in the reproduction of those images, but there is reason to question whether they in fact show the fungal haustorium without its cell wall. Considering the potential implications for interpreting the structure in the context of inter-symbiont transfer, further investigation is needed. In the present work, we examine the symbiont contact zone in Endocarpon pusillum Hedw., a well-known member of the Verrucariaceae (Eurotiomycetes), and compare it with divergent examples from two isidiose Porina species (Gyalectales; Lecanoromycetes) and several foliicolous lichens (Lecanorales) showing different interface morphologies and degrees of intraparietal penetration and wall reduction. We attempt to determine whether haustorial walls are absent, reduced, or reinforced in these taxa, and consider the implications for the functional significance of lichen haustoria in their diverse forms.
Methods and Materials
Lichen samples were collected from south-western Florida, USA (Calopadia puiggarii (Müll. Arg.) Vězda, Endocarpon pusillum, Porina microcoralloides Ertz et al. and P. nanoarbuscula Ertz et al.), and Tenerife (Byssoloma kakouettae (Sérus.) Lücking & Sérus., B. leucoblepharum (Nyl.) Vain. and Fellhanera bouteillei (Desm.) Vězda), Spain, in the course of other studies focused on these taxa (Sanders et al. Reference Sanders, Pérez-Ortega, Nelsen, Lücking and de los Ríos2016, Reference Sanders, De Carolis, Ertz, de los Ríos and Muggia2023; Sanders & de los Ríos Reference Sanders and de los Ríos2017, Reference Sanders and de los Ríos2023).
Fresh material, misted in Petri plates with distilled water 24 h previously, was hand-sectioned with a thin razor blade. Sections were placed immediately into ice-chilled tubes with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.1) for c. 3 h, then washed in buffer three times in 3 h, post-fixed with 1% osmium tetroxide (5 h), washed again, then dehydrated in a graded ethanol series (25-35-50-75-95-100(×3)%) followed by propylene oxide. Specimens were next infiltrated with Spurr’s low viscosity resin (initially diluted with propylene oxide) for 72 h and polymerized at 60 °C (de los Ríos & Ascaso Reference de los Ríos, Ascaso, Kranner, Beckett and Varma2002). Specimen blocks were sectioned with an Ultracut ultramicrotome, stained with uranyl acetate and lead citrate, and imaged with a JEOL JEM 1400 Flash transmission electron microscope.
Results
Endocarpon pusillum and Diplosphaera phycobiont
Earliest indications of intraparietal penetration appeared as a heterogeneous area of disruption within the algal cell wall that soon acquired electron density (Fig. 1A & B, horizontal arrow). The adjacent fungal cell wall showed discontinuity in the outer layers (Fig. 1A & C, double arrow) while inner wall layers expanded into the gap (Fig. 1A, oblique arrows) and became thickened (Fig. 1C). The fungal cell wall layers, especially the inner ones, were highly electron-transparent. Later stages showed the thickened protrusion of fungal wall material projecting further into the zone of algal wall degradation in front of it (Fig. 1D & E, Fig. 2A). Lomasome-like vesicular and tubular membrane modifications were present in the cytoplasm of both symbionts adjacent to the zone of incipient penetration (Figs 1A, 2A, 3A, arrowheads). Abundant lomasomes were also seen in symbiont cells at zones of contact where penetration pegs were not evident in those planes of section (Figs 2 & 3, arrowheads). In Fig. 1E, a concentrically folded membranous structure was positioned in the fungal cytoplasm directly behind the wall protrusion. In Fig. 3A & B, a larger, clearly membrane-bounded vesicle or plasmalemma fold appeared just outside the fungal protoplast immediately adjacent to the wall protrusion. A similar structure, separated within the plane of section from the protoplast by a thin bridge of wall material, was also seen in an algal cell adjacent to fungal cell contact where the algal wall appeared thickened (Fig. 3E & F). Such structures were observed repeatedly. The boundary between contacting fungal and algal cell walls often had a sinuous, corrugated appearance (Fig. 3C & D). Not infrequently, wall material from both symbionts appeared to intermix irregularly, making the exact boundary between fungal and algal cell walls unclear (Fig. 3A).
Figure 1. TEM images of symbiont contact zones between Endocarpon pusillum (f) and its phycobiont Diplosphaera (a). Developmental stages of intraparietal haustorium formation. A, initiation of haustorium from new inner layer of fungal cell wall (oblique arrows) protruding through break in outer wall layers (double arrows). Single horizontal arrow indicates zone of algal cell wall dissolutions. Note lomasome-like membranous inclusions (arrowheads), vesicular in fungal cell and tubular in algal cell, exterior to respective plasmalemmas and continuous with cell wall materials. B, protrusion into area of algal wall disruption (single horizontal arrow); arrowheads indicate lomasome-like membranous inclusions. C, thickening of incipient penetration peg emerging from rupture in outer fungal cell wall layers (double arrows). D & E, further intrusion and thickening of incipient haustorium into area of algal cell wall dissolution (horizontal arrow). Note continuity of peg base with innermost layers of fungal cell wall (oblique arrows in E). In E, plasmalemmasome-like folds of concentric membranes (m) are positioned just behind the haustorium within the fungal cell. Abbreviations: a = algal cell; f = fungal cell; aw = algal cell wall; fw = fungal cell wall; m = plasmalemmasome-like concentric membranous folds; p = fungal wall protrusion/peg-like haustorium; v = vacuole. Scales: A–E = 200 nm.
Figure 2. TEM images of symbiont contact zones between Endocarpon pusillum (f) and its phycobiont Diplosphaera (a). Abundant vesicular and tubular inclusions (arrowheads) between algal plasmalemma and cell wall, also in fungal cell in C; note thickening of algal wall. Horizontal arrow in A indicates area of algal cell wall dissolution. Abbreviations: a = algal cell; f = fungal cell; aw = algal cell wall; fw = fungal cell wall; p = peg-like intraparietal haustorium. Scales: A–C = 200 nm.
Figure 3. TEM images of symbiont contact zones between Endocarpon pusillum (f) and its phycobiont Diplosphaera (a). A, broad boundary showing two fungal pegs to the right and highly thickened, indistinctly intermixed fungal and algal wall materials to the left. Horizontal arrows indicate region of algal cell wall degradation ahead of fungal penetration pegs, directly behind which an exterior elaboration of the fungal plasmalemma is evident (asterisk). Arrowheads indicate smaller, lomasome-like vesicles/tubules emerging from algal plasmalemma and embedded within wall material. B, asterisk indicates elaboration of the fungal plasmalemma similar to the previous micrograph; the material separating it from the rest of the protoplast is clearly continuous with and part of the fungal cell wall. C & D, sinuous contact zones (arrows) between fungal and algal walls. Arrowheads indicate lomasome-like vesicles and tubules between protoplast and cell wall of algal symbiont in contact region. E, asterisk indicates exterior elaboration of algal protoplast, similar to that shown for the fungus in A and B, with a thin layer of wall material clearly separating it from rest of protoplast, at least within this plane of the section. Arrowheads indicate lomasome-like vesicles and tubules between the protoplast and cell wall of both symbionts in the contact area. F, formation of bridging wall material (arrow) similar to that shown in previous micrograph, in longitudinal view or in process of development. Abbreviations: a = algal cell; f = fungal cell; aw = algal cell wall; fw = fungal cell wall; p = peg-like intraparietal haustorium. Scales: A & B = 100 nm; C–F = 200 nm.
Microfruticose/isidiose crustose Porina microcoralloides and its Trentepohlia phycobiont
Symbiont contact zones often consisted of a broad protuberance of the fungal cell that made a concave deformity in the surface of the algal symbiont cell (Fig. 4A, C & D). Reduction of the algal cell wall was moderate (Fig. 4A & D) to substantial (Fig. 4C). The fungal cell wall, elsewhere several hundred nm in thickness, was reduced to less than c. 50 nm at the contact zone (Fig. 4C & D), and in some instances could scarcely be distinguished (Fig. 4B). The fungal wall protrusion arose from inner wall layers that emerge through a break in the outer wall layers (Fig. 4C, double arrow). At some interfaces, the fungal cell surface was itself concave where the contacted algal symbiont protruded into the fungal cell (Fig. 4B), exactly reversing the expected positional roles of the two symbionts. As with the fungal haustorium, the algal protrusion was continuous with and clearly arose from the innermost wall layer(s) that appeared to have been laid down secondarily (Fig. 4B) and had broken through the outer layers.
Figure 4. TEM images of contact zones between lichen-forming fungus Porina microcoralloides (f) and its algal symbiont Trentepohlia (a). In A, C and D, a broad, thin-walled bulge in the fungal cell produces a concave deformation in the algal cell surface, with thinning of the algal cell wall (especially in C and D), while in B the algal symbiont protrudes into the fungal cell, whose wall is reduced to a faint remnant within the zone of contact. Note algal cell protrusion arising from inner wall layer (aw2). Arrowheads indicate remnant of fungal cell wall surrounding algal protrusion; arrows in C & D indicate fungal plasmalemma. Double arrows in C indicate rupture in outer fungal cell wall as inner layer emerges as penetrative bulge. Abbreviations: a = algal cell; f = fungal cell; aw = algal cell wall; fw = fungal cell wall. Scales: A = 500 nm; B = 1 μm; C & D = 250 nm.
Microfruticose/isidiose Porina nanoarbuscula and its Trentepohlia phycobiont
At symbiont contact zones, the fungal cell often showed a haustorium-like protuberance of variable size that produced a concave deformity in the cell surface of the contacted alga (Fig. 5A–C). However, in some cases the fungal contact surface was concave (Fig. 5D), with the cell wall highly reduced in thickness at the contact zone, often to the point of indistinguishability. Algal cell walls were also often reduced at the contact zone (Fig. 5B & C).
Figure 5. TEMs of contact zones between lichen-forming fungus Porina nanoarbuscula (f) and its algal symbiont Trentepohlia (a). Arrowheads indicate remnant fungal cell wall. Abbreviations: aw = algal cell wall; fw = fungal cell wall. Scales: A & C = 250 nm; B & D = 500 nm.
Crustose foliicolous Byssoloma kakouettae and its trebouxiophycean phycobiont
The fungal cell as a whole pushed broadly into the algal cell, which it often deformed into a crescent-shaped outline in section (Fig. 6A & C). There was no distinctive structure that could be recognized as a haustorium. At the contact zone, the fungal cell wall was reduced to no more than 50–100 nm in thickness (Fig. 6B–D). The contacted algal cell wall also showed a reduction in thickness.
Figure 6. TEM images of symbiont contacts between the lichen-forming fungus Byssoloma kakouettae (f) and its trebouxiophycean algal symbiont (a). B shows the contact zone of A at higher magnification. Arrowheads indicate remnant fungal cell wall; arrow in B indicates fungal cell plasmalemma. Abbreviations: aw = algal cell wall; fw = fungal cell wall. Scales: A = 1 μm; B–D = 200 nm.
Other crustose foliicolous lichens
Byssoloma leucoblepharum cells bulged into a broad concavity made in the contacted cells of its trebouxiophycean phycobiont. The fungal cell wall was sometimes reduced to the point of indistinguishability in the contact zone; deposition of algal wall materials in apposition to the protrusion was evident (Fig. 7A). Calopadia puiggarii cells protruded broadly against those of it phycobiont Heveochlorella (Fig. 7B); its cell wall was reduced to less than 100 nm in the contact zone. Fellhanera bouteillei, in chiefly appositional contact with its phycobiont Chloroidium, showed a reduction of its cell wall to a thickness of c. 50 nm (Fig. 7C & D).
Figure 7. TEM images of symbiont contacts between three foliicolous lichen-forming fungi and their ‘Watanabealean’ (Trebouxiophyceae) algal symbionts. A, Byssoloma leucoblepharum. B, Calopadia puiggarii. C & D, Fellhanera bouteillei. Arrowheads indicate remnant fungal cell wall; arrows indicate fungal cell plasmalemma. Abbreviations: aw = algal cell wall; fw = fungal cell wall. Scales: A, B & D = 500 nm; C = 200 nm.
Discussion
Haustorial penetration into the algal cell lumen was not seen in any of the microlichens examined in the present study, including the foliicolous taxa that have little or no differentiation of thallus tissue layers (Sanders & de los Ríos Reference Sanders and de los Ríos2016, Reference Sanders and de los Ríos2023). All exhibited intraparietal penetrative contacts between symbionts, although of quite varied morphologies, ranging from narrow, peg-like wall protrusions to broad, undifferentiated portions of the fungal cell surface. The former are comparable to the intraparietal haustoria described in other lichen-forming taxa, while the latter invaginate or deform the phycobiont cell directly without elaboration of any distinctive penetrative structure. Degradation of the algal cell wall at the symbiont interface, with concomitant thinning of the fungal cell wall, qualifies these broad contacts as intraparietal even though no differentiated haustorium can be recognized. Studies of plant pathogenic fungi have likewise reported a wide variety of invasive contacts, for which the definition of haustorium was found to be insufficient to encompass the range of structures to which this term has been applied (Calonge Reference Calonge1969).
The haustoria we observed in Endocarpon pusillum resemble one depicted in a previous ultrastructural survey of that taxon (Ahmadjian & Jacobs Reference Ahmadjian and Jacobs1970). They are minute, intraparietal pegs formed from a newer, interior fungal wall layer(s) that breaks through the outer layers, as observed in other lichen-forming taxa such as the basidiolichen genus Dictyonema (Oberwinkler Reference Oberwinkler, Schwemmler and Schenk1980). In our observations, these haustoria are always enclosed within a fungal cell wall. The inner wall layer from which the haustorium arises is relatively thin initially, but soon thickens, particularly at the haustorial tip (Figs 1C–E, 3A). Vacuoles are usually present in the vicinity, and one referee of this manuscript suggested the possibility that they might be involved in generating the turgor pressure for wall protrusion. We find the intraparietal haustorium of E. pusillum to be quite similar morphologically to those observed in other Verrucariaceae, such as Dermatocarpon miniatum (L.) W. Mann (Galun et al. Reference Galun, Kushnir, Behr and Ben-Shaul1973; figs 1–7) and Verrucaria spp. (Kushnir & Galun Reference Kushnir and Galun1977; figs 1–5), although we disagree with those authors’ interpretation of the haustorium as a naked protoplast. The frequent presence of plasmalemmasomes, lomasomes or lomasome-like vesicles and tubules localized at contact zones in cells of both symbionts is suggestive of active construction and/or modification of cell walls. Lomasomes were first noted in early TEM studies of fungi as plasmalemma-derived elaborations exterior to the cell and associated with cell wall materials (Girbardt Reference Girbardt1958). They were also observed in similar contexts within green algal cells (Barton Reference Barton1965; Crawley Reference Crawley1965), and in the lichen-forming fungus Peltigera canina (Boissière Reference Boissière1982; Lallemant et al. Reference Lallemant, Boissière, Boissière, Leclerc, Velly and Wagner1986). The term plasmalemmasome has also been applied to folded modifications of the cell membrane. According to Marchant & Moore (Reference Marchant and Moore1973), plasmalemmasomes differ from lomasomes in that the former are not surrounded by wall materials. Although certain membrane elaborations observed in TEM have been shown to be artifacts of chemical fixation and processing (e.g. Büdel & Rhiel Reference Büdel and Rhiel1987), lomasomes and plasmalemmasomes are evident in cryofixed as well as chemically processed samples, and therefore appear to be genuine features of living cells (Marchant & Moore Reference Marchant and Moore1973). Their precise functions have not been clarified but seem to be associated with wall growth and modification (Wilsenach & Kessel Reference Wilsenach and Kessel1965). They may also play a role in the recycling or removal of excess membrane involved in such growth processes (Mazheika et al. Reference Mazheika, Psurtseva and Kamzolkina2022), which may add more membrane to the plasmalemma through exocytosis than can be accommodated by the rate of cell enlargement (Riquelme et al. Reference Riquelme, Aguirre, Bartnicki-García, Braus, Feldbrügge, Fleig, Hansberg, Herrera-Estrella, Kämper and Kück2018). In Endocarpon, the visible integration of lomasome-like membranous structures among the innermost layers of wall material in contacting cells of both symbionts (Figs 1A & B, 2, 3A), and the larger, single elaborations of the plasmalemma bridged by material clearly continuous with the cell wall (Fig. 3A, B, E & F) suggest a role in cell wall construction. Vesicle remnants within the mycobiont cell wall have also been noted (Büdel & Rhiel Reference Büdel and Rhiel1987) and may represent a subsequent stage of this process. The membranous elaborations are particularly evident where cell wall thickening is visibly taking place. Deposition of encapsulating algal wall material in apposition to fungal penetrative structures has been observed at contact interfaces in many other lichen taxa (Geitler Reference Geitler1934; Tschermak Reference Tschermak1941; Honegger Reference Honegger1984), as well as plant hosts of fungal pathogens (Bracker & Littlefield Reference Bracker, Littlefield, Byrde and Cutting1973). The contacting cells at the symbiont interface in Endocarpon thus appear to be at least as much involved in wall deposition as they are in its degradation. The thickening of the short, narrow, intraparietal peg, as well as the algal cell wall in apposition to it, are not suggestive of adaptations likely to accelerate solute transfer across the contact interface. From a phylogenetic perspective, such interfaces might be interpreted as arising from a dynamic equilibrium between fungal invasion and algal defence that stabilizes the lichen symbiosis, as previous authors have proposed. Haustorial penetration of algal prey, a pre-symbiotic means of obtaining resources from a victimized cell, may have lost its original function when the need for extractive organs was obviated by the evolution of algal carbohydrate release. A separate but equally interesting question is whether those peculiar peg-like intrusions also have some functional significance in their present form.
We propose that such structures may serve an important function in firmly anchoring the symbiont cells to each other at their contact zones. As poikilohydric partnerships, lichens typically undergo daily cycles of hydration and desiccation, with concomitant extremes of swelling and shrinkage in tissues and cells. These forces can easily rupture tissues, which in some cases contributes significantly to lichen morphogenesis. Examples include the tissue perforation in the lace lichen (Lutz Reference Lutz1894; Peirce Reference Peirce1898; Sanders Reference Sanders1989) and fissuring of the epinecral layer in Lasallia (Sanders & de los Ríos Reference Sanders and de los Ríos2018), as well as the characteristic cracking of the cortex in many saxicolous crusts (Brodo et al. Reference Brodo, Sharnoff and Sharnoff2001; Sanders et al. Reference Sanders, Moe and Ascaso2004), particularly evident in the splitting of thallus units into secondary areolae (Létrouit-Galinou & Asta Reference Létrouit-Galinou and Asta1994). At the cellular level, such stresses risk pulling fungal and algal cells apart at their junctions, potentially rupturing the layer of hydrophobic sealant that canalizes solute transfer between them. In the intertidal lichen Wahlenbergiella tavaresiae (R.L. Moe) Gueidan et al. (also a member of the Verrucariaceae), the intraparietal haustorium bears lateral flanges that are embedded within the wall of the phycobiont like the barbs of a spear, ensuring that symbiont cells are held together in close contact (Sanders et al. Reference Sanders, Moe and Ascaso2004). In Endocarpon, contacting walls of fungal and algal symbionts are often interlocked in sinuous, corrugated borders (Fig. 3C & D), also evident in a published micrograph of endolithic Verrucaria sp. (Kushnir & Galun Reference Kushnir and Galun1977; fig. 5). In other places, mutual construction or reconstruction intermeshes fungal and algal cell walls such that the exact boundary between them is not readily distinguishable (Fig. 3A). These features suggest further adaptations to integrate adjacent symbiont cells in resistance to separating stresses. Malachowski et al. (Reference Malachowski, Baker and Hooper1980) viewed the peg-like intraparietal haustoria they observed in Usnea cavernosa as serving to ‘hold algal cells close as thallus matures and air spaces develop’. That haustoria can effectively maintain physical unification of symbiont cells is corroborated by the observations of Honegger (Reference Honegger and Brown1985). She reported that Coccomyxa algae, which are completely surrounded but not penetrated by hyphae of their Peltigera and Solorina partners, can be easily separated from the mycobiont in the laboratory, whereas no pure fraction of phycobionts can be obtained from lecanoralean mycobionts that penetrate their trebouxioid algal symbionts with intracellular or intraparietal haustoria. In lichens with autospore-forming Trebouxia as phycobiont, fungal penetration and separation of dividing algal cells is thought to play a mechanical role in distributing algal symbionts in coordination with mycobiont growth (Greenhalgh & Anglesea Reference Greenhalgh and Anglesea1979; Honegger Reference Honegger1987).
Benefiting in part from the improved resolution in electron microscopy over recent decades, our interpretation of the intraparietal haustorium found in Endocarpon and Verrucariaceae differs from that offered in previous works (Galun et al. Reference Galun, Paran and Ben-Shaul1971b, Reference Galun, Kushnir, Behr and Ben-Shaul1973; Kushnir & Galun Reference Kushnir and Galun1977; Galun Reference Galun and Galun1988). On the other hand, the extreme level of wall thinning we observed in other taxa suggests that a naked haustorial protoplast in lichen-forming fungi may not be a far-fetched idea. In the microfruticose/isidiose Porina spp. and the foliicolous taxa examined (Figs 4–7), the cell wall of the fungal protrusion was often substantially reduced to no more than 50 to 100 μm in thickness, and in many instances was hardly distinguishable (Figs 4B & C, 5B–D, 6C & D, 7A). However, the protrusion was always in direct contact with the algal cell wall, with no space visible between them. The actual thickness of the fungal cell wall is likely to be even less than that estimated from micrographs, since only those sections that are exactly perpendicular to the wall will display its true thickness, while oblique sections will exaggerate it. Somewhat peg-like protrusions were occasionally observed in these lichens (Fig. 7D), but in general their symbiont interfaces tended to involve broader surfaces without differentiated haustoria. These more extensive contact surfaces, in combination with the highly reduced cell walls, appear more likely to facilitate solute exchange than the peg-like haustoria, and would seem somewhat less suited to maintaining physical unity of symbiont cells. Before interpreting the highly thinned walls as any sort of adaptation, however, one should keep in mind that the protrusion is initiated from an inner wall layer that, at least initially, will be only a fraction of the wall’s total thickness.
Between lichen symbionts, protrusion and penetration can be reciprocal (Figs 4B & 5D). Geitler (Reference Geitler1933) noted a counter-intrusion by cyanobiont cells into the appressoria of Lempholemma chalazanum (Ach.) B. de Lesd., which he interpreted as an indication that the alga fights back against fungal attack. Finger-like projections observed in cyanobiont cells squeezed free of mycobiont hyphae in Thyrea pulvinata (Schaer.) A. Massal. (Geitler Reference Geitler1936; fig. 4h) probably also correspond to such reciprocal intrusions of the algal symbiont into the fungal partner. In a published TEM micrograph, the Gloeocapsa-like symbiont of Gonohymenia mesopotamica J. Steiner can be seen penetrating a cell of the mycobiont (Paran et al. Reference Paran, Ben-Shaul and Galun1971; fig. 1, lower right), although it is not commented upon in the text. Penetrative reciprocity was noted in contact zones between the intertidal lichen-forming fungus Wahlenbergiella tavaresiae and its phaeophycean phycobiont Petroderma, which showed interdigitating haustoria-like interpenetrations, with concomitant thinning of both algal and fungal cell walls (Sanders et al. Reference Sanders, Moe and Ascaso2004; figs 40–42). As with their mycobionts, the algal symbionts Petroderma and Trentepohlia are filamentous, that is, with polar growth in a linear trajectory that can be directed intrusively. Recent genomic studies in trebouxiophycean phycobionts describe genes coding for enzymes that digest fungal wall polymers such as lichenan, which the authors speculate might be used by the alga as an additional source of glucose (Puginier et al. Reference Puginier, Libourel, Otte, Škaloud, Haon, Grisel, Petersen, Berrin, Delaux and Dal Grande2024). Such results at least suggest an enzymatic basis for reciprocal penetration. If it seems surprising that the alga should be stimulated to grow into fungal cells, we should recall that maintaining a functional contact interface is also of importance to the algal partner. Yet the current system of classifying symbiont contacts in lichens (appositional, intraparietal, intracellular) only takes into account the degree to which the algal cell wall is breached, while ignoring any dissolution of that of the mycobiont. To a large extent this reflects the expectation that the mycobiont will play an offensive role and the algal symbiont a defensive one at the cellular level. This longstanding idea may require some reconsideration.
Acknowledgements
We are deeply grateful to the staff of the electron microscopy facility at the Centro Nacional de Biotecnología (CNB–CSIC), Madrid, and especially to its current director, Beatriz Martín Jouve, and her predecessor, Cristina Patiño, for repeatedly accommodating our projects with their highly skilled preparation of ultrathin sections and technical support for TEM imaging. Financial support for microscopy work was provided by the grant PID2019-105469RB-C22 (AEI, MICIU). The manuscript benefited from critical review by two anonymous referees.
Author ORCIDs
William B. Sanders, 0000-0001-9572-4244; Asunción de los Ríos Murillo, 0000-0002-0266-3516.
Competing Interests
The authors declare none.
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