Borderline lichen

The concept of the borderline lichen emerged from long-standing debates over what constitutes a lichen in systematic treatments of lichenized fungi.^[1] Historically, many definitions centred on the presence of a thallus: a vegetative body composed of fungal and algal components differing from either partner grown alone.^[2] A widely used definition proposed by Hawksworth in 1988 described a lichen as a stable, self-supporting association of a mycobiont and a photobiont in which the mycobiont is the "exhabitant" (the exterior layer) and the alga an interior but extracellular inhabitant.^[3]

Borderline cases challenged this model because their fungal partners may be intermixed with, or even within, the photobiont's tissues rather than clearly forming an exterior layer.^[4] In a marine context, Kohlmeyer and co-authors used "borderline lichens" for loose, often submerged associations in which the fungal partner does not develop a well-differentiated cortex and is not clearly exhabitant under prevailing definitions.^[4][3] They argued that these associations should still be recognized as lichens because they show specialization involving derived ascomycete orders and because the fungus modifies the algal partner's morphology.^[4] In 2001, separate groups led by Kováčik and Pereira and by Lud and co-authors independently investigated the developmental morphology of polar maritime species and concluded that simple organizational levels did not preclude lichen status.^[5][6] Shortly afterwards, the term "optional lichenization" was introduced by Wedin and co-authors in 2004 for fungi that switch between borderline lichenized and saprotrophic modes depending on the substrate.^[7]

In a broader synthesis, Lücking and co-authors noted that some lichen-like associations fit the mycobiont–photobiont framework but remain unstable, show little morphogenetic effect, or blur with other categories such as lichenicolous fungi on sterile thalli. They emphasized morphogenesis (a novel morphology not found in the separate bionts) and the photobiont body plan as useful criteria for delimiting borderline and marginal cases.^[1] Sanders reviewed these conceptual boundaries, noting that while the "exhabitant" model (where the fungus forms an exterior layer) is standard, many lichens are structurally simpler, especially species growing within bark or rock where the fungus may simply contact algal surfaces without forming specialized tissues.^[8] The term "primitive lichen" appears in some older literature as a near-synonym, but modern lichenology generally prefers "borderline" to describe a boundary between different structural and nutritional strategies rather than implying an evolutionary hierarchy.^[9]

The primary characteristic of borderline lichens is the absence of a structured, stratified thallus.^[4][9] In complex lichens, the fungus builds specialized tissues such as an upper and lower cortex, which protect the photobiont from desiccation and excessive light. The internal structure is typically heteromerous (layered).^[2] Borderline lichens lack these features. Instead of forming plectenchyma (dense, tissue-like structures formed by fused hyphae), the fungus maintains an irregular, filamentous network that interweaves with the photosynthetic colonies.^[9][11] In marine borderline lichens, the partners may show specialization and altered photobiont morphology, yet still lack the well-differentiated fungal tissues often treated as characteristic of lichens (except in reproductive structures such as ascomata).^[4]

These associations sit between better-defined categories. They differ from mycophycobioses—associations where a fungus lives within an alga without altering its form—because the fungus shows specialization and the photobiont shows noticeable structural changes.^[4] What separates borderline lichens from simple fungal infestations is the morphogenetic influence of the mycobiont: the fungal partner triggers changes in the arrangement, division, or morphology of the algal cells.^[4][5] In the association between Mastodia tessellata and Prasiola, for example, the fungus causes the foliose (leaf-like) alga to separate into packets of cells, a pattern not seen in free-living Prasiola.^[6]

Borderline lichens often lack the diverse secondary metabolites found in macrolichens. Kohlmeyer and co-authors noted that the Mastodia–Prasiola association does not form secondary lichen substances, consistent with reduced lichen-like organization in the vegetative thallus.^[4] However, the fungal partner still produces characteristic reproductive structures such as perithecia, apothecia, and spermogonia, which may be well-developed even when the vegetative thallus is absent or inconspicuous.^[4][9]

Borderline lichens represent a polyphyletic assemblage of fungi that have independently evolved symbiotic lifestyles.^[9] Recent analyses suggest that the lichen-forming lifestyle evolved at least 20 to 30 times independently in more than five different classes of Ascomycota and several orders of Basidiomycota.^[8] Borderline and optional lichenization are described as scattered across distant fungal groups, including marine-intertidal lineages such as Collemopsidium and substrate-plastic lineages in Stictidaceae.^[12][9][11]

In many cases, borderline lichens represent single species within genera or families that are otherwise composed of non-lichenized, saprotrophic, or parasitic fungi, suggesting that the transition to a lichenized mode of nutrition can occur with minimal structural modification.^[7][9] Grube and Wedin interpreted such facultative, substrate-dependent lichenization as a present-day analogue of early stages of thallus evolution, before more advanced forms of organization arose.^[9] Muggia and co-authors similarly argued that studying simple borderline forms by observation and experiment may inform understanding of how fungal–algal interactions transition from loose contact to stable, structured lichen symbioses.^[13]

A significant development in the classification of borderline lichens occurred in 2016 with the establishment of the order Collemopsidiales within the clade Dothideomyceta.^[12] Pérez-Ortega and co-authors described this order to accommodate the family Xanthopyreniaceae, which includes the marine borderline-lichen genus Collemopsidium and the lichenicolous genus Zwackhiomyces.^[12] Using five fungal fossils as calibration points (including Paleopyrenomycites devonicus), they inferred a crown age of approximately 230 million years for Collemopsidiales, placing its diversification in the Triassic, shortly after the Permian–Triassic extinction event.^[12] The exact phylogenetic position of the family within the Dothideomyceta remains uncertain: six molecular markers were insufficient to assign it with certainty to either Arthoniomycetes or Dothideomycetes.^[12]

The family Stictidaceae illustrates the fluid boundary between saprotrophic and borderline lichenized lifestyles.^[7] Some species in this family exhibit optional lichenization, switching from a saprotrophic mode on dead wood to a borderline lichenized mode associating with Coccomyxa algae on bark, depending on the substrate.^[7] Ancestral character state analysis for this group indicates that a saprotrophic ancestor was most likely, with lichenization occurring independently in various lineages, suggesting that the evolutionary step between these fungal lifestyles is smaller than previously anticipated.^[7][9]

The genus Collemopsidium (family Xanthopyreniaceae) comprises marine borderline lichens that typically associate with filamentous cyanobacteria of the genus Hyella.^[12][14] Rocky seashores are the primary habitat: species in this genus inhabit the midlittoral to supralittoral zones and frequently grow as endoliths, boring into calcareous rocks or the shells of marine organisms.^[12][14] Their thalli are often entirely immersed within the substrate, with only the carbonised, black perithecia visible as minute dots on rock or shell surfaces.^[12]

A 2016 study using two molecular markers (nrLSU and mtSSU) and the general mixed Yule–coalescent model (GMYC) established that the global diversity of marine Collemopsidium is far greater than previously recognized: approximately 26 putative species were inferred worldwide, compared with six morphological species recognized previously in Europe.^[12][14] Bayes factor comparison strongly supported the higher-diversity model.^[12] The study also found that rock-boring ability has been acquired and lost multiple times throughout the evolutionary history of the group, indicating that this trait evolved in parallel in different lineages within Collemopsidiales.^[12] The maritime borderline lichen Collemopsidium pelvetiae, which grows on the brown seaweed Pelvetia canaliculata, was also treated by Kohlmeyer and co-authors as a borderline lichen example.^[4]

In a cave on the Coastal Range of the Atacama Desert in Chile, the extremely halotolerant black yeast Hortaea werneckii has been found growing among colonies of the microalga Dunaliella atacamensis in an association reminiscent of borderline lichens.^[13] D. atacamensis is the only member of the genus Dunaliella reported from a subaerial habitat, where it persists as non-motile colonies surrounded by a gelatinous matrix (a palmella stage) with thickened cell walls.^[13] The cave is inhabited by spiders whose silk threads collect condensing fog, supporting the growth of adhering algal colonies among which melanised cells of H. werneckii are frequently observed.^[13] Co-cultivation experiments using different growth media and cultivation approaches (including solid and liquid media, alginate inclusions, and dialysis membranes) have failed to show clear mutual effects between the two species in a laboratory setting.^[13] D. atacamensis could not be grown in vitro despite repeated attempts, and on solid media H. werneckii grew mostly in its yeast form, with melanised cells detected as dark spots within the algal colonies.^[13] The authors discussed this negative experimental outcome in the context of how fungal–algal interactions might transition from loose contact to stable, structured lichen symbioses, noting that the association may still be in a very early evolutionary stage and that more stressful conditions might be needed to trigger lichenization.^[13]

The relationship between the ascomycete Mastodia tessellata (a member of the Verrucariaceae, a family consisting mainly of lichen-forming fungi in terrestrial and intertidal habitats)^[8] and the foliose green alga Prasiola is one of the most widely studied borderline systems.^[4] Found in maritime polar regions such as the Antarctic Peninsula, the association was variously classified as a mycophycobiosis or fungal infestation because the fungal partner lives within the algal tissue, complicating the distinction between exhabitant and inhabitant.^[5][15] Kohlmeyer and co-authors reported that the mycelium forms a dense network of interwoven hyphae around algal cells without a differentiated cortex layer and noted the absence of secondary lichen substances.^[4]

Independent developmental studies by Lud and co-authors and by Kováčik and Pereira concluded that, despite its simple organizational level, the association qualifies as a lichen because the fungus decisively alters the arrangement of the algal cells, which become separated by gelatinised hyphae.^[5][6] The photobiont provides carbon to the fungus, while the symbiosis increases the alga's tolerance of freezing temperatures.^[6] Using multilocus sequence data (nrITS, RPL10A, and the plastid gene tufA) and species delimitation analyses, Garrido-Benavent and co-authors proposed that Mastodia tessellata associates with at least two species of Prasiola across its bipolar range, including P. borealis and an undescribed lineage restricted to the Antarctic Peninsula.^[15]

Schizoxylon albescens (family Stictidaceae) is a boreal ascomycete that exemplifies optional lichenization.^[7][11] On bark of Populus tremula (aspen), the fungus forms a borderline lichen morph: white patches containing minute fruiting bodies approximately 0.5–1 mm across, surrounded by clumps of the green alga Coccomyxa.^[11] On dead aspen wood, the same species occurs as a saprotrophic morph, producing slightly larger fruiting bodies (approximately 1–2 mm) without lichenized patches.^[7][11] Using high-throughput sequencing of the bacterial 16S rRNA gene together with fluorescence in situ hybridisation, Fernández-Brime and co-authors reported that bacterial communities differed between the two morphs but were determined chiefly by substrate (bark versus wood) rather than by the presence of algal photobionts.^[11] They concluded that the simple organization of borderline lichenization in Schizoxylon lacks the complex thallus structure required to host the highly specific microbiota typical of advanced lichens.^[11]

Trizodia acrobia is a microscopic ascomycete that forms a tripartite association with cyanobacterial colonies (mostly Nostoc) and peat mosses, and has been described as a borderline lichen.^[9][16][17] The fungus produces translucent white to cream-coloured apothecia up to 0.24 mm in diameter on the basal leaves of apical Sphagnum branches, specifically where dark green spots of Nostoc occur on the moss heads.^[16][17] Its hyphae are extracellular on living Sphagnum cells but enter the water-filled, dead hyaline cells through cell pores to envelop cyanobacterial colonies both on the moss surface and inside the leaf, without forming an organized thallus.^[16][17] Stenroos and co-authors found the fungus in all 44 Sphagnum–Nostoc associations they examined, but never in Sphagnum populations where cyanobacteria were absent, suggesting obligate cyanotrophy combined with specificity to the moss substrate.^[16]

In a five-gene phylogenetic analysis of symbioses between ascomycetes and bryophytes, Stenroos and co-authors placed Trizodia as basal to the Leotiomycetes, representing a previously unrecognized lineage within the Ascomycota.^[16] The authors described the association as potentially a structurally undeveloped form of a bryosymbiotic cyanolichen, noting that it does not form organized thallus structures but shares several characteristics with lichens, including apparent dependence on its photobiont and a hemiamyloid ascus wall.^[16]