{{Short description|Loosely organized fungal–photobiont symbioses}} {{Use Oxford spelling|date=March 2026}}
'''Borderline lichen''' is a term used in [[lichenology]] for structurally simple, [[symbiosis|symbiotic]] associations between a [[fungus]] and a [[photosynthesis|photosynthetic]] partner (an [[alga]] or [[cyanobacterium]]). Unlike typical [[lichen]]s, these associations lack a differentiated [[thallus]] (vegetative body) and a protective outer [[cortex (botany)|cortex]]. Instead, the fungus forms a loose network of [[mycelia]] intertwined with the photosynthetic cells. A defining feature is that the fungal partner triggers visible changes in the arrangement or form of the photobiont's cells, distinguishing borderline lichens from simple fungal colonization. The term was introduced in 2004 by Kohlmeyer, [[David Leslie Hawksworth|Hawksworth]], and Volkmann-Kohlmeyer for certain marine and maritime associations, including ''[[Mastodia tessellata]]'' and ''[[Collemopsidium pelvetiae]]'', that resemble lichens yet lack the organized fungal tissues expected under standard lichen definitions. The concept has since been extended to include fungi that switch between free-living and loosely lichenized modes depending on their [[substrate (biology)|substrate]] (termed "optional lichenization"), [[bryophyte]]-associated cyanobacterial systems in which fungal–photobiont contact occurs without organized thallus development, and associations between stress-tolerant fungi and algae in nutrient-poor environments such as caves in the [[Atacama Desert]].
Borderline lichens do not form a single evolutionary group. Instead, they represent a [[polyphyletic]] assemblage of fungi from distantly related lineages that have independently evolved symbiotic lifestyles. They have been proposed as present-day analogues of early stages of [[thallus]] evolution, before more complex lichen organization arose. Their simple structure has also made them a focus of debate over whether the lichen symbiosis is best understood as a [[mutualism (biology)|mutualism]] or as a form of controlled [[parasitism]], because the costs and benefits to each partner are considered more readily observable than in structurally complex lichens. These associations are often described as occupying the transitional zone between free-living organisms and fully integrated lichen symbioses.
==Terminology and conceptual origin==
The concept of the borderline lichen emerged from long-standing debates over what constitutes a [[lichen]] in systematic treatments of lichenized fungi.<ref name="Lucking et al. 2016"/> 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.<ref name="Hawksworth & Honegger 1994"/> A widely used definition proposed by [[David Leslie Hawksworth|Hawksworth]] in 1988 described a lichen as a stable, self-supporting association of a {{lichengloss|mycobiont}} and a {{lichengloss|photobiont}} in which the mycobiont is the "exhabitant" (the exterior layer) and the alga an interior but extracellular inhabitant.<ref name="Hawksworth 1988"/>
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.<ref name="Kohlmeyer et al. 2004"/> 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 {{lichengloss|cortex}} and is not clearly exhabitant under prevailing definitions.<ref name="Kohlmeyer et al. 2004"/><ref name="Hawksworth 1988"/> They argued that these associations should still be recognized as lichens because they show specialization involving derived [[Ascomycota|ascomycete]] orders and because the fungus modifies the algal partner's [[morphology (biology)|morphology]].<ref name="Kohlmeyer et al. 2004"/> 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.<ref name="Lud et al. 2001"/><ref name="Kovacik & Pereira 2001"/> Shortly afterwards, the term "optional lichenization" was introduced by Wedin and co-authors in 2004 for fungi that switch between borderline lichenized and [[saprotroph]]ic modes depending on the [[substrate (biology)|substrate]].<ref name="Wedin et al. 2004"/>
In a broader synthesis, [[Robert Lücking|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.<ref name="Lucking et al. 2016"/> 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.<ref name="Sanders 2023"/> 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.<ref name="Grube & Wedin 2016"/>
{| class="wikitable" |+ Key terminology in borderline lichenology ! Term !! Year !! Proposed/popularised by !! Context |- | [[Mycophycobiosis]] || 1972 || Kohlmeyer and Kohlmeyer<ref name="Kohlmeyer & Kohlmeyer 1972"/> || Fungi living inside multicellular algae without morphological change |- | Exhabitant definition || 1988 || Hawksworth<ref name="Hawksworth 1988"/> || Fungal partner as the outer layer of the symbiosis |- | Borderline lichen || 2004 || Kohlmeyer, Hawksworth and Volkmann-Kohlmeyer<ref name="Kohlmeyer et al. 2004"/> || Specialised loose symbioses with modified algal morphology |- | Optional lichenization || 2004 || Wedin, Döring and Gilenstam<ref name="Wedin et al. 2004"/> || Fungi switching between saprotrophy and loose lichenization |}
==Diagnostic characteristics==
The primary characteristic of borderline lichens is the absence of a structured, stratified thallus.<ref name="Kohlmeyer et al. 2004"/><ref name="Grube & Wedin 2016"/> 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 {{lichengloss|heteromerous}} (layered).<ref name="Hawksworth & Honegger 1994"/> Borderline lichens lack these features. Instead of forming {{lichengloss|plectenchyma}} (dense, tissue-like structures formed by fused [[hypha]]e), the fungus maintains an irregular, filamentous network that interweaves with the photosynthetic colonies.<ref name="Grube & Wedin 2016"/><ref name="Fernandez-Brime et al. 2019"/> 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 [[ascoma]]ta).<ref name="Kohlmeyer et al. 2004"/>
These associations sit between better-defined categories. They differ from [[mycophycobiosis|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.<ref name="Kohlmeyer et al. 2004"/> 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.<ref name="Kohlmeyer et al. 2004"/><ref name="Lud et al. 2001"/> 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''.<ref name="Kovacik & Pereira 2001"/>
Borderline lichens often lack the diverse [[secondary metabolite]]s found in macrolichens. Kohlmeyer and co-authors noted that the ''Mastodia''–''Prasiola'' association does not form secondary [[lichen substance]]s, consistent with reduced lichen-like organization in the vegetative thallus.<ref name="Kohlmeyer et al. 2004"/> However, the fungal partner still produces characteristic reproductive structures such as [[perithecia]], [[apothecia]], and {{lichengloss|spermogonia}}, which may be well-developed even when the vegetative thallus is absent or inconspicuous.<ref name="Kohlmeyer et al. 2004"/><ref name="Grube & Wedin 2016"/>
{| class="wikitable" |+ Comparison of structural traits: borderline vs. stratified lichens ! Feature !! Borderline lichen !! Stratified/macrolichen |- | Thallus cortex || Absent or poorly developed || Well-differentiated and {{lichengloss|conglutinated}} |- | Internal stratification || Absent ({{lichengloss|homoiomerous}}) || Distinct layers ({{lichengloss|heteromerous}}) |- | Fungal organization || Loose mycelia || Complex {{lichengloss|plectenchyma}} |- | Secondary chemistry || Limited; mostly [[melanin]] || Diverse (acids, [[depside]]s, [[depsidone]]s)<ref name="Lucking et al. 2016"/> |- | Substrate penetration || Often {{lichengloss|endolithic}} or {{lichengloss|endophloeodal}} || Typically {{lichengloss|epilithic}} or {{lichengloss|epiphytic}} |}
==Evolutionary origins and classification== [[File:2015-08-10 Collemopsidium foveolatum (A.L. Sm.) F. Mohr 545932.jpg|thumb|right|alt=Close-up of barnacle shells; many tiny black dots (perithecia) are scattered across the pale calcareous surface |Intertidal barnacle shells colonized by ''Collemopsidium foveolatum'', visible as pepper-like black perithecia]] Borderline lichens represent a [[polyphyletic]] assemblage of fungi that have independently evolved symbiotic lifestyles.<ref name="Grube & Wedin 2016"/> 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]].<ref name="Sanders 2023"/> Borderline and optional lichenization are described as scattered across distant fungal groups, including marine-intertidal [[lineage (evolution)|lineages]] such as ''[[Collemopsidium]]'' and substrate-plastic lineages in [[Stictidaceae]].<ref name="Perez-Ortega et al. 2016"/><ref name="Grube & Wedin 2016"/><ref name="Fernandez-Brime et al. 2019"/>
In many cases, borderline lichens represent single [[species]] within [[genera]] or [[Family (taxonomy)|families]] that are otherwise composed of non-lichenized, [[saprotroph]]ic, or [[parasitic fungi]], suggesting that the transition to a lichenized mode of nutrition can occur with minimal structural modification.<ref name="Wedin et al. 2004"/><ref name="Grube & Wedin 2016"/> 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.<ref name="Grube & Wedin 2016"/> 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.<ref name="Muggia et al. 2020"/>
===The order Collemopsidiales===
A significant development in the classification of borderline lichens occurred in 2016 with the establishment of the order [[Collemopsidiales]] within the clade [[Dothideomyceta]].<ref name="Perez-Ortega et al. 2016"/> 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]]''.<ref name="Perez-Ortega et al. 2016"/> Using five fungal fossils as calibration points (including ''[[Paleopyrenomycites devonicus]]''), they inferred a [[crown group|crown]] age of approximately 230 million years for Collemopsidiales, placing its diversification in the [[Triassic]], shortly after the [[Permian–Triassic extinction event]].<ref name="Perez-Ortega et al. 2016"/> The exact phylogenetic position of the family within the Dothideomyceta remains uncertain: six [[molecular marker]]s were insufficient to assign it with certainty to either [[Arthoniomycetes]] or [[Dothideomycetes]].<ref name="Perez-Ortega et al. 2016"/>
===Transitions from saprotrophy===
The family Stictidaceae illustrates the fluid boundary between saprotrophic and borderline lichenized lifestyles.<ref name="Wedin et al. 2004"/> 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.<ref name="Wedin et al. 2004"/> [[Ancestral reconstruction|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.<ref name="Wedin et al. 2004"/><ref name="Grube & Wedin 2016"/>
==Example systems==
===''Collemopsidium'' species===
The genus ''[[Collemopsidium]]'' (family [[Xanthopyreniaceae]]) comprises marine borderline lichens that typically associate with filamentous [[cyanobacteria]] of the genus ''[[Hyella]]''.<ref name="Perez-Ortega et al. 2016"/><ref name="Mohr et al. 2004"/> Rocky seashores are the primary habitat: species in this genus inhabit the midlittoral to [[supralittoral zone]]s and frequently grow as [[endolith]]s, boring into [[calcareous]] rocks or the shells of marine organisms.<ref name="Perez-Ortega et al. 2016"/><ref name="Mohr et al. 2004"/> Their thalli are often entirely immersed within the substrate, with only the {{lichengloss|carbonised}}, black [[perithecium|perithecia]] visible as minute dots on rock or shell surfaces.<ref name="Perez-Ortega et al. 2016"/>
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.<ref name="Perez-Ortega et al. 2016"/><ref name="Mohr et al. 2004"/> Bayes factor comparison strongly supported the higher-diversity model.<ref name="Perez-Ortega et al. 2016"/> 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.<ref name="Perez-Ortega et al. 2016"/> 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.<ref name="Kohlmeyer et al. 2004"/>
===''Hortaea werneckii'' and ''Dunaliella atacamensis''=== [[File:Hortaea-werneckii-fungus--causes-tinea-nigra.jpg|thumb|alt=Light micrograph showing long, branching, dark filaments with several clustered, darker cell masses|Microscopic view of the halotolerant black yeast ''Hortaea werneckii'', one of the partners proposed for a borderline lichen-like association with the microalga ''Dunaliella atacamensis'' in Atacama Desert caves]] In a cave on the Coastal Range of the [[Atacama Desert]] in Chile, the extremely [[halotolerance|halotolerant]] black yeast ''[[Hortaea werneckii]]'' has been found growing among [[colony (biology)|colonies]] of the microalga ''[[Dunaliella atacamensis]]'' in an association reminiscent of borderline lichens.<ref name="Muggia et al. 2020"/> ''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 wall]]s.<ref name="Muggia et al. 2020"/> 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.<ref name="Muggia et al. 2020"/> Co-cultivation experiments using different [[growth media]] and cultivation approaches (including solid and liquid media, [[alginate]] inclusions, and [[dialysis (chemistry)|dialysis]] membranes) have failed to show clear mutual effects between the two species in a laboratory setting.<ref name="Muggia et al. 2020"/> ''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.<ref name="Muggia et al. 2020"/> 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.<ref name="Muggia et al. 2020"/>
===''Mastodia tessellata'' and ''Prasiola''=== [[File:Mastodia y Papúa.jpg|thumb|right|alt=A shoreline boulder with irregular black, crusty growth and scattered yellow-green patches; a rocky beach, grey slope, and penguins are blurred in the background|Coastal rock covered with dark colonies of ''Mastodia tessellata'' at the shoreline; penguins stand on the beach in the background]] The relationship between the ascomycete ''Mastodia tessellata'' (a member of the [[Verrucariaceae]], a family consisting mainly of lichen-forming fungi in terrestrial and [[intertidal zone|intertidal]] habitats)<ref name="Sanders 2023"/> and the foliose green alga ''[[Prasiola]]'' is one of the most widely studied borderline systems.<ref name="Kohlmeyer et al. 2004"/> 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.<ref name="Lud et al. 2001"/><ref name="Garrido-Benavent et al. 2017"/> 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.<ref name="Kohlmeyer et al. 2004"/>
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.<ref name="Lud et al. 2001"/><ref name="Kovacik & Pereira 2001"/> The photobiont provides carbon to the fungus, while the symbiosis increases the alga's tolerance of freezing temperatures.<ref name="Kovacik & Pereira 2001"/> Using multilocus sequence data ([[internal transcribed spacer|nrITS]], [[60S ribosomal protein L10a|RPL10A]], and the [[plastid]] gene [[EF-Tu|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.<ref name="Garrido-Benavent et al. 2017"/>
===''Schizoxylon albescens'' and optional lichenization===
''[[Schizoxylon albescens]]'' (family [[Stictidaceae]]) is a [[boreal ecosystem|boreal]] ascomycete that exemplifies optional lichenization.<ref name="Wedin et al. 2004"/><ref name="Fernandez-Brime et al. 2019"/> On bark of ''[[Populus tremula]]'' (aspen), the fungus forms a borderline lichen [[Polymorphism (biology)|morph]]: white patches containing minute [[fruiting bodies]] approximately 0.5–1 mm across, surrounded by clumps of the green alga ''[[Coccomyxa]]''.<ref name="Fernandez-Brime et al. 2019"/> On dead aspen wood, the same species occurs as a saprotrophic morph, producing slightly larger fruiting bodies (approximately 1–2 mm) without lichenized patches.<ref name="Wedin et al. 2004"/><ref name="Fernandez-Brime et al. 2019"/> Using [[high-throughput sequencing]] of the bacterial [[16S ribosomal RNA|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.<ref name="Fernandez-Brime et al. 2019"/> 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.<ref name="Fernandez-Brime et al. 2019"/>
===''Trizodia acrobia''===
''[[Trizodia acrobia]]'' is a microscopic ascomycete that forms a tripartite association with [[cyanobacterium|cyanobacterial]] colonies (mostly ''[[Nostoc]]'') and [[Sphagnum|peat mosses]], and has been described as a borderline lichen.<ref name="Grube & Wedin 2016"/><ref name="Stenroos et al. 2010"/><ref name="Eckstein 2021"/> 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.<ref name="Stenroos et al. 2010"/><ref name="Eckstein 2021"/> Its hyphae are extracellular on living ''Sphagnum'' cells but enter the water-filled, dead [[hyalocyte|hyaline cells]] through cell pores to envelop cyanobacterial colonies both on the moss surface and inside the leaf, without forming an organized thallus.<ref name="Stenroos et al. 2010"/><ref name="Eckstein 2021"/> [[Soili Stenroos|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.<ref name="Stenroos et al. 2010"/>
In a [[molecular phylogenetics|five-gene phylogenetic analysis]] of symbioses between ascomycetes and [[bryophyte]]s, Stenroos and co-authors placed ''Trizodia'' as [[basal (phylogenetics)|basal]] to the [[Leotiomycetes]], representing a previously unrecognized lineage within the [[Ascomycota]].<ref name="Stenroos et al. 2010"/> 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 [[amyloid (mycology)|hemiamyloid]] ascus wall.<ref name="Stenroos et al. 2010"/>
==Symbiotic function and the mutualism debate==
The nature of the interaction in borderline lichens is central to broader debates about whether the lichen symbiosis should be viewed as a [[mutualism (biology)|mutualism]] or as a more one-sided relationship.<ref name="Sanders 2023"/> Because borderline associations lack the complex protective structures of macrolichens, the costs and benefits of the relationship are sometimes considered more readily observable.<ref name="Grube & Wedin 2016"/>
===Carbon transfer===
A hallmark of lichenization is the mass transfer of photosynthetically fixed [[carbohydrate]]s from the photobiont to the mycobiont.<ref name="Sanders 2023"/> In eukaryotic algal lichens, this transfer involves [[sugar alcohol]]s ([[ribitol]], [[erythritol]], or [[sorbitol]], depending on the genus), whereas [[cyanobiont]]s transfer glucose; the mycobionts convert the sugars received into [[mannitol]] and [[arabitol]].<ref name="Sanders 2023"/> Sanders noted that these carbon transfer systems show remarkable convergences across independently evolved lichen lineages, and that when a compatible fungus is encountered, the alga proactively releases large amounts of carbohydrate in response to symbiotic signalling, a process that ceases when the alga is isolated into culture.<ref name="Sanders 2023"/> This active release, rather than forcible extraction, suggests that the alga is a participant in the partnership rather than a passive victim. Whether the same transfer mechanisms operate in borderline lichens, where contact between partners is less intimate, has not been directly tested.<ref name="Sanders 2023"/>
===Benefits and protections===
Borderline lichens provide evidence of environmental protection for the photobiont in stressful [[microhabitat]]s. In the ''Mastodia''–''Prasiola'' system, the alga's tolerance of freezing is enhanced in symbiosis.<ref name="Kovacik & Pereira 2001"/> In maritime environments, the rock-boring mycelia of borderline lichens provide a stable, moisture-retaining refuge for cyanobacteria that might otherwise be desiccated or washed away.<ref name="Perez-Ortega et al. 2016"/> More generally, Sanders argued that lichen-forming fungi conserve rather than consume their algal symbionts, and that in the stressful environments where lichens are successful, the mutual self-interests of both partners substantially align.<ref name="Sanders 2023"/>
===Parasitic interpretations===
Despite these observations, some prominent biologists have questioned whether the algal partner truly benefits. [[Vernon Ahmadjian]], for example, characterized the relationship as "controlled parasitism" rather than mutualism, while others point to the proactive release of carbohydrates by the algae as evidence of a cooperative partnership.<ref name="Sanders 2023"/> Borderline lichens are often cited in this debate because their lack of complex structure makes the immediate costs and benefits to each partner easier to observe than in macrolichens.<ref name="Grube & Wedin 2016"/> In some borderline cases, the dynamics between partners can appear antagonistic. In the leaf-dwelling ''[[Strigula]]'', [[Harry Marshall Ward|Ward]] (1884) observed that if the mycelium encounters a [[germination|germinating]] spore or few-celled germling of its algal partner ''[[Cephaleuros]]'', the alga is overpowered and destroyed. Ward further suggested that a stable equilibrium can be established only when the fungus contacts a well-established alga.<ref name="Sanders 2023"/> Sanders argued, however, that the proactive release of carbohydrate by the alga, the generally healthy appearance of algal cells within lichen thalli, and the substantial alignment of both partners' long-term interests under stressful conditions are not easily reconciled with a purely parasitic interpretation.<ref name="Sanders 2023"/>
===Microbiota===
Because borderline lichens lack much of the differentiated thallus structure that in typical lichens forms a long-lived microhabitat, they have been examined to test how morphological organization relates to associated microbiota.<ref name="Fernandez-Brime et al. 2019"/><ref name="Grube & Wedin 2016"/> In the ''Schizoxylon'' system, [[microbial consortium|bacterial communities]] in lichenized and saprotrophic morphs were found to be distinct but determined chiefly by substrate rather than by the algal photobiont, suggesting that a more complex thallus is required for hosting specific microbial communities.<ref name="Fernandez-Brime et al. 2019"/> Grube and Wedin noted that the recognition of additional microbial associates in lichen thalli, including bacteria and yeasts, has complicated traditional two-partner views of the symbiosis and contributed to renewed debate over lichen delimitation.<ref name="Grube & Wedin 2016"/>
==Research approaches==
Research on borderline lichens draws on microscopy, [[molecular phylogenetics]], and culture-based experiments, often in combination.<ref name="Kohlmeyer et al. 2004"/><ref name="Fernandez-Brime et al. 2019"/><ref name="Perez-Ortega et al. 2016"/><ref name="Muggia et al. 2020"/> [[Light microscopy]] is widely used for describing ascoma anatomy, and Kohlmeyer and co-authors used line drawings to document and clarify the nomenclature of ''Mastodia'' and ''Collemopsidium''.<ref name="Kohlmeyer et al. 2004"/> Because many borderline lichens are immersed within their substrates, specialized imaging is often required. Pérez-Ortega and co-authors used [[scanning electron microscopy]] in [[backscatter|backscattered]] electron mode (SEM-BSE) to explore fine-scale interactions between endolithic fungi and calcareous shells,<ref name="Perez-Ortega et al. 2016"/> while Fernández-Brime and co-authors applied [[fluorescence in situ hybridisation]] combined with [[confocal laser scanning microscopy]] (FISH-CLSM) to localize bacterial communities within the loose mycelia of the ''Schizoxylon'' system.<ref name="Fernandez-Brime et al. 2019"/>
[[Molecular phylogenetics]] and species delimitation methods have revealed hidden diversity within borderline lichen groups. Pérez-Ortega and co-authors combined multi-locus phylogenetic analyses with GMYC-based species delimitation to resolve lineages within Collemopsidiales,<ref name="Perez-Ortega et al. 2016"/> while Garrido-Benavent and co-authors applied multilocus sequence analyses to explore species boundaries among the ''Prasiola'' photobionts associated with ''Mastodia tessellata''.<ref name="Garrido-Benavent et al. 2017"/> Culture-based experiments complement these molecular approaches. Muggia and co-authors conducted co-cultivation trials with ''Hortaea werneckii'' and ''Dunaliella'' under multiple media and cultivation conditions to test whether the two organisms would develop lichen-like organization when grown together.<ref name="Muggia et al. 2020"/>
==References== <references>
<ref name="Eckstein 2021">{{cite journal |last=Eckstein |first=Jan |title=''Trizodia acrobia'', ein mit Cyanobakterien assoziierter Ascomycet auf Torfmoosen |trans-title=''Trizodia acrobia'', an ascomycete associated with cyanobacteria on peat moss |journal=Boletus |volume=42 |issue=1 |year=2021 |pages=53–55 |language=de}}</ref>
<ref name="Fernandez-Brime et al. 2019">{{cite journal |last1=Fernández-Brime |first1=Samantha |last2=Muggia |first2=Lucia |last3=Maier |first3=Stefanie |last4=Grube |first4=Martin |last5=Wedin |first5=Mats |title=Bacterial communities in an optional lichen symbiosis are determined by substrate, not algal photobionts |journal=FEMS Microbiology Ecology |volume=95 |issue=3 |year=2019 |article-number=fiz012 |doi=10.1093/femsec/fiz012 |doi-access=free}}</ref>
<ref name="Garrido-Benavent et al. 2017">{{cite journal |last1=Garrido-Benavent |first1=Isaac |last2=Pérez-Ortega |first2=Sergio |last3=de los Ríos |first3=Asunción |title=From Alaska to Antarctica: species boundaries and genetic diversity of ''Prasiola'' (Trebouxiophyceae), a foliose chlorophyte associated with the bipolar lichen-forming fungus ''Mastodia tessellata'' |journal=Molecular Phylogenetics and Evolution |volume=107 |year=2017 |pages=1–13 |doi=10.1016/j.ympev.2016.10.013}}</ref>
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</references>
[[Category:Lichenology]] [[Category:Symbiosis]] [[Category:Fungal morphology and anatomy]]