William Bennett Sanders

Lichen-forming fungi of genus Botryolepraria build no compact thalli, yet elevate and display algal symbionts upon their open, aerial mycelium. Although Botryolepraria occurs worldwide, the construction of its unique somatic form has not been examined in detail. We applied light microscopy and SEM to better understand how it is built and stabilized and how phycobionts are distributed during development.PREMISELichen-forming fungi of genus Botryolepraria build no compact thalli, yet elevate and display algal symbionts upon their open, aerial mycelium. Although Botryolepraria occurs worldwide, the construction of its unique somatic form has not been examined in detail. We applied light microscopy and SEM to better understand how it is built and stabilized and how phycobionts are distributed during development.Specimens were examined with light microscopy, conventional SEM, and cryo-field emission SEM. Symbiont identity was corroborated by obtaining and comparing nucleotide sequences with those in the NCBI database.METHODSSpecimens were examined with light microscopy, conventional SEM, and cryo-field emission SEM. Symbiont identity was corroborated by obtaining and comparing nucleotide sequences with those in the NCBI database.Hyphal branches grew centripetally toward clusters of algal symbionts, while other branches grew centrifugally outward before further bifurcating to produce additional hyphal branches that reoriented centripetally toward algal clusters. Anastomosis of hyphae, tip to tip or laterally via short bridging connections, occurred frequently. The lichen was irregularly but often densely covered with thread-like hydrophobic materials that resemble certain forms of plant epicuticular waxes. Repeated interpenetration of suspended algal clusters by anastomosing mycobiont hyphae separated and distributed phycobiont cells within the expanding reticulum. Fungal ITS and LSU and algal rbcL sequences suggest closest proximity of mycobiont and phycobiont to Botryolepraria neotropica and Pseudostichococcus monallantoides, respectively, for the material studied.RESULTSHyphal branches grew centripetally toward clusters of algal symbionts, while other branches grew centrifugally outward before further bifurcating to produce additional hyphal branches that reoriented centripetally toward algal clusters. Anastomosis of hyphae, tip to tip or laterally via short bridging connections, occurred frequently. The lichen was irregularly but often densely covered with thread-like hydrophobic materials that resemble certain forms of plant epicuticular waxes. Repeated interpenetration of suspended algal clusters by anastomosing mycobiont hyphae separated and distributed phycobiont cells within the expanding reticulum. Fungal ITS and LSU and algal rbcL sequences suggest closest proximity of mycobiont and phycobiont to Botryolepraria neotropica and Pseudostichococcus monallantoides, respectively, for the material studied.Anastomosis of hyphae, in regions where algae are absent and at the surfaces of expanding phycobiont clusters, stabilizes the soma of Botrylopraria as a three-dimensional lattice. The dense covering of hydrophobic materials over an open aerial mycelium suggests adaptation to avoid surface condensation and optimize gas exchange.CONCLUSIONSAnastomosis of hyphae, in regions where algae are absent and at the surfaces of expanding phycobiont clusters, stabilizes the soma of Botrylopraria as a three-dimensional lattice. The dense covering of hydrophobic materials over an open aerial mycelium suggests adaptation to avoid surface condensation and optimize gas exchange.

This work attempts to better understand the significance of morphological diversity among fungal-algal contact zones present in lichens. We used TEM to examine a variety of lichen symbioses involving non-trebouxialean green algae that show intraparietal penetration by the mycobiont. A principal focus was on Endocarpon pusillum , a well-known member of a family ( Verrucariaceae ; Eurotiomycetes ) previously reported to be characterized by unwalled haustoria exposing a naked fungal protoplast. Peg-like haustoria arose from an inner layer(s) of the mycobiont cell wall that broke through outer layers and penetrated a short distance into the wall of the green algal symbiont ( Diplosphaera ). In both fungal and algal cells at the contact interface, lomasome-like vesicles and tubules occurred as modifications of the plasmalemma intermixed with wall materials at the inner surface of the cell wall. A fungal cell wall was consistently present around the haustorium, which resembled those depicted in earlier TEM studies of Verrucariaceae. Previously published micrographs of Verrucariaceae purporting to show wall-less haustoria surrounded by an empty space are believed to have been misinterpreted. However, in the isidiose Porina and foliicolous Calopadia , Byssoloma and Fellhanera species ( Lecanoromycetes ), we did observe extreme degrees of reduction in the mycobiont cell wall at symbiont contact interfaces. In those lichens, a broad area of the fungal cell bulged into the adjacent algal symbiont, broadly invaginating the wall of the latter and penetrating it intraparietally without differentiation of a distinct haustorial structure. The mycobiont wall surrounding such protrusions often thinned to near indistinguishability towards its extremity. The protrusion made direct contact with the algal cell wall; no empty space occurred between them. We propose that the short, peg-like intraparietal haustoria bind the symbionts and help maintain cell contacts amid the stresses of tissue expansion and shrinkage, thereby avoiding disruption of the continuous hydrophobic coating that facilitates transfer between them. Broader contact interfaces with extremely thin adjacent walls may facilitate solute flow between symbionts. Reciprocal penetration of algal protrusions into mycobiont cells, noted in Porina as well as other lichens studied previously, is a neglected but potentially significant indication that both symbionts may actively work to maintain functional contact interfaces.

While the diversity of foliicolous lichen-forming fungi has been explored in substantial depth, relatively little attention has been paid to their algal symbionts. We studied the unicellular green phycobionts of the lecanoralean lichens Bacidina (Ramalinaceae), Byssoloma, Fellhanera and Tapellaria (Pilocarpaceae) and graphidalean Gyalectidium (Gomphillaceae) from two extratropical foliicolous communities in continental Spain and the Canary Islands. We examined the pyrenoids of algal symbionts within thalli using TEM, and obtained several algal nrSSU and rbcL sequences from whole thalli, and also from cultures isolated from some of these lichens. Pyrenoid structure and molecular sequence data provided support for recognizing Chloroidium (Watanabeales, Trebouxiophyceae) as phycobiont in thalli of Byssoloma subdiscordans and Fellhanera bouteillei (Pilocarpaceae) in both communities. Bacidina apiahica (Ramalinaceae) and Tapellaria epiphylla (Pilocarpaceae) likewise appeared to partner with Chloroidium based on the presence of the same pyrenoid type, although we were able to obtain a phycobiont sequence only from a culture isolate of the latter. These results contrast with those obtained previously from a foliicolous lichen community in southern Florida, which revealed only strains of Heveochlorella (Jaagichlorella) as phycobiont of foliicolous Pilocarpaceae and Gomphillaceae. On the other hand, the pyrenoid we observed in the phycobionts associated with Gyalectidium setiferum and G. minus corresponded to that of Heveochlorella (Jaagichlorella). However, the poor quality of the phycobiont sequence data obtained from G. minus, probably due to the presence of epibiontic algae, could not provide additional perspective on the pyrenoid structure observations. Nonetheless, clear differences in pyrenoid ultrastructure can allow Chloroidium and Heveochlorella phycobionts to be distinguished from each other in TEM. Our results indicate a greater diversity of unicellular green-algal symbionts in foliicolous communities from Spain than previously observed in other geographical areas, and suggest that further studies focused on symbiont pairing in these communities might reveal distinctive and varied patterns of phycobiont preference.

Excerpt: Lichen-forming fungi and their algal symbionts together produce composite thalli that often have the appearance and properties of unitary organisms. Although the exact contours of the concept may vary according to viewpoint, lichens have been commonly distinguished from other fungal–algal symbioses by a useful and widely cited definition (Hawksworth, 1988; Hawksworth & Honegger, 1994), wherein the mycobiont comprises the external component (exhabitant) that houses the interior but extracellular algal symbiont(s). More recently, increased attention has been focused on additional microorganisms, particularly fungi and bacteria, that form part of the lichen thallus microbiome (Grube et al., 2015; Spribille et al., 2016; Cernava et al., 2017; Smith et al., 2020; Tzovaras et al., 2020; Cometto et al., 2022). This has revealed a new dimension to lichen biology, similar to that recognizable in plants and animals as the diverse microbial inhabitants of their internal and external surfaces are explored (Porras-Alfaro & Bayman, 2011; Ezenwa et al., 2012; Gilbert et al., 2018). Such research highlights the degree to which biological communities coexist at very different scales. A plant, an animal, and a lichen thallus are all components of a broader community of interacting macro-organisms, while at a finer level of organization, each may individually harbor its own microbial community or communities. Although microbiome research has changed how we think about plants and animals, it has not resulted in any effort to redefine them. By contrast, a number of lichen biologists have recently proposed expanding the definition of a lichen to include other microorganisms that colonize the thallus. These proposals, and indeed the question of whether a new definition is warranted or desirable, ought to be considered critically.

Abstract Lichens are classic examples of symbiosis, but some biologists have questioned whether the algal partner benefits from the relationship. Among the diverse lichen symbioses, the carbon transfer systems show remarkable convergences. When a compatible fungus is encountered, the alga proactively releases large amounts of carbohydrate, suggesting active participation rather than victimhood. Some lichen-related fungus–alga symbioses appear obligatory for the algal partner. Within true lichens, algal symbionts can persist at microsites where they might not otherwise be competitive, because of improved stress tolerance, reduced photoinhibition, protection from herbivores, and the more efficient moisture management and positioning for light interception that fungal structures provide. Algal clones continually disperse from the lichen thallus by diverse means, allowing the genotype to pioneer aposymbiotic colonies from a stable refuge. Because lichen-forming fungi conserve rather than consume their algal symbionts, the mutual self-interests of both partners substantially align in the stressful microhabitats where lichens are successful.

Porina is a widely distributed, species-rich genus of crustose, lichen-forming fungi, some with thalline outgrowths that have been recognized as isidia. We studied three taxa with thalli consisting chiefly of ascending isidioid structures occurring on trunks and branches of Taxodium in southwestern Florida, and provide details of their structure with light and electron microscopy. Two of these taxa we describe as new species: P. microcoralloides and P. nanoarbuscula. Genetic sequences (mtSSU) suggest that they are closely related to each other, yet they differ markedly in the size, morphology and anatomical organization of their isidioid branches as well as in the length of their ascospores. In the three Floridian taxa studied, the crustose portion of the thallus is partly endophloeodic and partly superficial, the latter often patchy, evanescent or inconspicuous, and completely lacks the differentiated anatomical organization characteristic of the isidioid structures arising from it. In Porina microcoralloides, the ascendant thallus consists of branched, coralloid inflated structures with phycobiont (Trentepohlia) unicells arranged at the periphery of a loose central medulla. Sparse fungal cells are interspersed and overlie the algal layer in places, but no differentiated cortex is present, leaving phycobiont cells more or less exposed at the surface. In the closely related Porina nanoarbuscula, the isidioid structures are much finer, more densely branched, and composed of a single, central file of roughly spherical Trentepohlia cells surrounded by a jacket of subglobose fungal cells. The ascospores of P. microcoralloides are more than twice the length of those of P. nanoarbuscula. Although thalli of these two Porina species occur in the same habitats and are sometimes found growing alongside each other, phylogenetic analysis of rbcL sequences suggest that they partner with distinct clades of Trentepohlia phycobionts. A third taxon examined, Porina cf. scabrida, is morphologically rather similar to P. microcoralloides, but the ascendant branches are bright yellow-orange, more cylindrical, and corticated by a thin layer of agglutinated fungal hyphae; perithecia were not seen. Analysis of mtSSU sequences places it distant from P. microcoralloides and P. nanoarbuscula phylogenetically. None of the Floridian taxa studied was particularly close to the European isidiate species Porina hibernica and P. pseudohibernica, which appeared as sister to each other in the analysis. While a particular type of isidiose structure may be reliably characteristic of specific taxa, similarities or differences in these structures do not seem to be useful indicators of phylogenetic proximity or distances among taxa. The morphological trends evident in Porina suggest that multiple transitions from crustose to isidioid or microfruticose growth have arisen repeatedly and in quite different ways within this single genus. At least some of the diverse structures treated within the broad concept of isidia may be representative of the developmental pathways by which fruticose growth forms may arise.

Numerous distinct clades of lichen-forming fungi have independently specialized as foliicolous colonists of living leaves in the humid tropics and subtropics. Because of technical difficulties, the anatomy of their minute crustose thalli has not been compared in detail. In the present study, we applied SEM-BSE imaging to sectioned blocks of embedded thalli representing six lecanoralean taxa of foliicolous lichen-forming fungi with unicellular green algal partners. We compared our observations with those obtained in a previous study of foliicolous Gomphillaceae (Ostropales), which utilize a similar type of algal partner. The upper surface of the thalli was a mostly continuous layer of mycobiont hyphae of typical diameter, unlike the largely acellular epilayer found previously in the foliicolous Gomphillaceae. Byssoloma leucoblepharum was exceptional in lacking a covering layer altogether. Thalli were essentially unstratified, with algal symbionts not confined to any distinct layer. Whereas the prothallus of foliicolous Gomphillaceae was derived from the overlying epilayer, in the lecanoralean taxa examined here the prothallus was derived from hyphae continuous with either the upper surface of the thallus or the lower surface, or both. This finding suggests that the prothallus of lichen forming fungi may represent structures of developmentally different origins in different taxa.

Excerpt: The April issue of Nature Plants featured an editorial (Nature Photoaerogens?)1 addressing my proposal of the term photoaerogen to refer to cyanobacteria, eukaryotic algae and plants collectively. Of course I was thrilled that my essay2 received such high-profile attention. But I also felt that the editorial frequently misses the point. Its subtitle and first paragraph highlight, with amusing anecdotes, the difficulties and fundamental arbitrarity of classification systems. Yet my essay made no suggestions regarding classification or biosystematic nomenclature. Photoaerogen was explicitly proposed as a term without taxonomic implications. While some biological concepts group organisms biosystematically according to phylogenetic affinities, others recognize common functional or ecological properties regardless of phylogen.

The recently described genus Rhizonema is among the most important cyanobacterial partners in lichen symbioses, but its morphological characterization in the genus diagnosis - true branching of the T-type - appears at odds with several published figures showing false branching. We investigated cyanobiont branching and cell division with light microscopy in two basidiolichens from Florida and one from Japan, including aposymbiotically cultured material of the latter. Mycobiont species identities (Cyphellostereum jamesianum, Dictyonema darwinianum, and D. moorei) and photobiont genus identity (Rhizonema) were corroborated with ITS and rbcLX sequences, respectively. Single and paired false branching occurred commonly in all three strains examined. False branches developed adjacent to necridic cells or heterocytes, or by separation of vegetative cells at compression folds in the trichome. Non-transverse cell divisions, usually oblique, were observed in two of the three Rhizonema strains examined. T-type true branches sometimes arose from such divisions, although oblique growth from the branch cell often resulted in ambiguous branch junctions. Additionally, Y-type true branches appeared to grow from contorted filaments. In cultured material, a kind of pseudo-branch sometimes arose from single- or several-celled segments liberated from trichome apices. The segments attached secondarily to filaments and grew there as apparent branches. We conclude that Rhizonema is a genus of considerable morphological flexibility, with multiple modes of branching possible in a single strain. While true branching or non-transverse divisions, when observable, may help distinguish Rhizonema from the phenotypically similar Scytonema, false branching occurs commonly in both genera, and therefore cannot be used to distinguish them.