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Microalgae isolation and culture protocols: (a) Buellia zoharyi thallus fixation, (b) upper cortex removal, (c) and (d) microalgae capture, (e) well-developed microalgae culture on agar plate, (f) and (g) microalgae growth cultures in liquid medium, and (h) triple streaking allows unique colonies to be obtained which will grow as axenic liquid cultures. Steps (a-h) were performed inside a laminar flow cabinet.
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Microalgae of the genus Watanabea are widely distributed as free living in soil/subaerial and aquatic habitats. In this study, two Watanabea spp. were isolated from lichen thalli of Buellia zoharyi collected on biocrusts in the Iberian Peninsula and the Canary Islands. To ascertain their taxonomic position and phylogenetic relationships within the...
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... algae, fragments of other lichen species or infection by lichenicolous fungi) the lichen material was examined using a sterile blade. Superficial sterilization was performed following the Arnold et al. (2009) method. The already sterilized thalli were placed in a stereomicroscope inside a laminar flow cabinet and fixed using adhesive tape (Fig. 1a). The upper cortex was removed using the sterile blade; therefore, the phycobiont layer was exposed (Fig. 1b). Algae colonies were captured using a Ø 0.3-mm sterile needle and were deposited on BBM agar plates, which were stored in a growth chamber at 20 C under a 12 /12 h light : dark cycle (lighting conditions: 15 μmol m À2 s À1 ) for ...
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... examined using a sterile blade. Superficial sterilization was performed following the Arnold et al. (2009) method. The already sterilized thalli were placed in a stereomicroscope inside a laminar flow cabinet and fixed using adhesive tape (Fig. 1a). The upper cortex was removed using the sterile blade; therefore, the phycobiont layer was exposed (Fig. 1b). Algae colonies were captured using a Ø 0.3-mm sterile needle and were deposited on BBM agar plates, which were stored in a growth chamber at 20 C under a 12 /12 h light : dark cycle (lighting conditions: 15 μmol m À2 s À1 ) for two to three months (Fig. 1c,d). Next, welldeveloped colonies were transferred into BBM liquid medium using ...
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... cortex was removed using the sterile blade; therefore, the phycobiont layer was exposed (Fig. 1b). Algae colonies were captured using a Ø 0.3-mm sterile needle and were deposited on BBM agar plates, which were stored in a growth chamber at 20 C under a 12 /12 h light : dark cycle (lighting conditions: 15 μmol m À2 s À1 ) for two to three months (Fig. 1c,d). Next, welldeveloped colonies were transferred into BBM liquid medium using sterile tips (Fig. 1e,f). After growing, 150 μL of algae culture was sonicated for ten min to disgregate the microalgae colonies, and axenic cultures were obtained by streaking on BBM plates (Fig. ...
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... Algae colonies were captured using a Ø 0.3-mm sterile needle and were deposited on BBM agar plates, which were stored in a growth chamber at 20 C under a 12 /12 h light : dark cycle (lighting conditions: 15 μmol m À2 s À1 ) for two to three months (Fig. 1c,d). Next, welldeveloped colonies were transferred into BBM liquid medium using sterile tips (Fig. 1e,f). After growing, 150 μL of algae culture was sonicated for ten min to disgregate the microalgae colonies, and axenic cultures were obtained by streaking on BBM plates (Fig. ...
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... (lighting conditions: 15 μmol m À2 s À1 ) for two to three months (Fig. 1c,d). Next, welldeveloped colonies were transferred into BBM liquid medium using sterile tips (Fig. 1e,f). After growing, 150 μL of algae culture was sonicated for ten min to disgregate the microalgae colonies, and axenic cultures were obtained by streaking on BBM plates (Fig. ...
Citations
... The identity of photobionts has only been studied in approximately 5% of lichens up to now [1,4,5]. Significant progress has been made in recent years with the description of several new genera and species of photobionts [6][7][8][9][10][11], suggesting that the true diversity of photobionts may be much greater than previously thought [4,5]. ...
This study explores the diversity of photobionts associated with the Mediterranean lichen-forming fungus Cladonia subturgida. For this purpose, we sequenced the whole ITS rDNA region by Sanger using a metabarcoding method for ITS2. A total of 41 specimens from Greece, Italy, France, Portugal, and Spain were studied. Additionally, two specimens from Spain were used to generate four cultures. Our molecular studies showed that the genus Myrmecia is the main photobiont of C. subturgida throughout its geographic distribution. This result contrasts with previous studies, which indicated that the main photobiont for most Cladonia species is Asterochloris. The identity of Myrmecia was also confirmed by ultrastructural studies of photobionts within the lichen thalli and cultures. Photobiont cells showed a parietal chloroplast lacking a pyrenoid, which characterizes the species in this genus. Phylogenetic analyses indicate hidden diversity within this genus. The results of amplicon sequencing showed the presence of multiple ASVs in 58.3% of the specimens studied.
... The investigated strain, ASUV135, from the Symbiotic Algal collection at the Universitat de València (ASUV, Spain), was isolated using the protocol described in Chiva et al. [27] from a thallus of the lichen Buellia zoharyi [23]. In the latter study [23], the isolated strain ASUV135 was identified by ITS rDNA phylogeny as a microalga of the genus Diplosphaera, named Diplosphaera sp. ...
... Three algal loci were amplified; the nuclear SSU rDNA was amplified using the primers 18F and 18R [33] and the nuclear ITS rDNA, including ITS1, 5.8S rDNA and ITS2, using the primer pair nr-SSU-1780 [34] and ITS 4T [35]; and the plastidial rbcL was amplified using the primers rbcL151f and rbcL986R [36]. PCR reactions for SSU and ITS were performed as described in Chiva et al. [27] and for rbcL, as described in Nelsen et al. [36]. Amplifications were carried out on a 96-well SensoQuest Labcycler (Progen Scientific). ...
Lichen phycobiomes have recently emerged as a source of biodiversity and new species of microalgae. Although in the genus Diplosphaera free-living microalgae are common, numerous strains belonging to this genus have frequently been recognized or isolated from lichen thalli. In this study, a comprehensive analysis of the strain Diplosphaera sp. ASUV135, isolated from a lichen thal-lus, has been carried out using an integrative taxonomic approach. The SSU and nuclear-encoding ITS rDNA, as well as the chloroplast rbcL gene, were sequenced and analyzed to ascertain its taxo-nomic position and phylogenetic relationships within the genus Diplosphaera. This strain was also analyzed by light, confocal and transmission microscopy for morphological and ultrastructural characterization. The phenotypic plasticity in this strain was also confirmed by changes in its morphology under different growth conditions, as well as those of modulated Chlorophyll a fluores-cence emissions, to understand its photosynthetic functioning. Our results pointed out that this strain represents a new taxon within the genus Diplosphaera (Prasiola group), described here as Diplo-sphaera 1longate sp. nova. This study also provides tools for future research on organisms with high phenotypic plasticity by using molecular, morphological, ultrastructural and physiological approaches .
... The lichen material was cleaned using a sterile blade to remove visible surface contamination, and it was then superficially sterilized following the Arnold et al. (2009) method. Two different protocols were used to isolate microalgae from these lichen fragments: a) the micro method described by Gasulla et al. (2010) was used to isolate microalgae from the thalli of R. farinacea and P. pseudotinctorum, and the resulting algal suspension was spread using the triple streak method on sterile 1.5% agar Bold's Basal Media Petri dishes (BBM) (Bold 1949;Bischoff and Bold 1963); and b), the method described in Chiva et al. (2021) was used on B. zoharyi thalli, which involved capturing tiny clumps of the algal layer using a Ø 0.3 mm sterile needle and inoculating them directly onto BBM. The isolated microalgae were maintained under a 50 µmol/ m − 2s − 1 photosynthetic photon flux density (PPFD) with a 12 h photoperiod at 18 °C. ...
... ITS4T (Kroken and Taylor 2000). PCR reactions for ITS loci were performed following Chiva et al. (2021). PCR products were visualized on 2% agarose gels and purified using the Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK). ...
... However, total sterilization of the surface cannot be guaranteed in lichens which are very close to the substrate. Surface cleaning is common both in studies related to the isolation and description of new species (e.g., Watanabea lichenicola Chiva, Dumitru, Bordenave & Barreno in Chiva et al. 2021) and in diversity and metabarcoding analyses in which symbiosis-associated microorganisms are the target, and assume the surface sterilization process as key to ensure that the results obtained come exclusively from organisms located inside the lichen thalli (Arnold et al. 2009;Muggia et al. 2013). In addition to surface sterilization, with the isolation protocol of Chiva et al. (2021) algae are captured from areas of the algal layer that have been removed with a sterile scalpel from the upper cortex. ...
The term phycobiome was recently introduced to designate all the microalgae (primary or non-primary) associated with
lichen symbioses. Abundant non-primary symbiotic microalgae are usually obtained from lichen isolations, confirming that
thalli are a source of biodiversity and new species. In this study, microalgae were isolated from thalli of Buellia zoharyi,
Ramalina farinacea and Parmotrema pseudotinctorum collected in the Iberian Peninsula and the Canary Islands. Excluding
Trebouxia phycobionts, 17 strains similar to Stichococcus (Prasiola clade) were obtained. Molecular identification was
carried out by nuclear ITS sequencing, and a phylogenetic tree was generated from these sequences, and grouping them
into 4 clades: Diplosphaera chodatti, Diplosphaera sp.1. Deuterostichocuccus sp.1. and Tritostichococcus coniocybes. It is
also noteworthy that Diplosphaera sp.1 was detected and isolated from three phylogenetically distant lichenized fungi (B.
zoharyi, R. farinacea and P. pseudotinctorum), which were sampled in ecologically different localities, namely Tenerife,
La Gomera and Castellón. These results reinforce the idea of the constant presence of certain microalgae associated with
the lichen thalli which, despite not being the main primary photobiont, probably form part of the lichen’s phycobiomes.
... Pure cultures of symbiotic algae are essential for species delimitation following an integrative taxonomic approach in which different analytical methods, combining morphology and genetic diversity, are considered [16,26,31,83,89,90]. Indeed, particularly for the genus Trebouxia, the lack of axenically cultured species has been, thus far, one of the major reasons for the unbalanced proportion between the genetically identified specieslevel lineages and the formally described species [5,6,15,16]. ...
Two microalgal species, Trebouxia jamesii and Trebouxia sp. TR9, were detected as the main photobionts coexisting in the thalli of the lichen Ramalina farinacea. Trebouxia sp. TR9 emerged as a new taxon in lichen symbioses and was successfully isolated and propagated in in vitro culture and thoroughly investigated. Several years of research have confirmed the taxon Trebouxia sp. TR9 to be a model/reference organism for studying mycobiont–photobiont association patterns in lichen symbioses. Trebouxia sp. TR9 is the first symbiotic, lichen-forming microalga for which an exhaustive characterization of cellular ultrastructure, physiological traits, genetic and genomic diversity is available. The cellular ultrastructure was studied by light, electron and confocal microscopy; physiological traits were studied as responses to different abiotic stresses. The genetic diversity was previously analyzed at both the nuclear and organelle levels by using chloroplast, mitochondrial, and nuclear genome data, and a multiplicity of phylogenetic analyses were carried out to study its intraspecific diversity at a biogeographical level and its specificity association patterns with the mycobiont. Here, Trebouxia sp. TR9 is formally described by applying an integrative taxonomic approach and is presented to science as Trebouxia lynnae, in honor of Lynn Margulis, who was the primary modern proponent for the significance of symbiosis in evolution. The complete set of analyses that were carried out for its characterization is provided.
Lichen symbiosis is centered around a relationship between a fungus and a photosynthetic microbe, usually a green alga. In addition to their main photosynthetic partner (the photobiont), lichen symbioses can contain additional algae present in low abundance. The biology of these algae and the way they interact with the rest of lichen symbionts remains largely unknown. Here we present the first genome sequence of a non-photobiont lichen-associated alga. Coccomyxa viridis was unexpectedly found in 12% of publicly available lichen metagenomes. With few exceptions, members of the Coccomyxa viridis clade occur in lichens as non-photobionts, potentially growing in thalli endophytically. The 45.7 Mbp genome of C. viridis was assembled into 18 near chromosome-level contigs, making it one of the most contiguous genomic assemblies for any lichen-associated algae. Comparing the C. viridis genome to its close relatives revealed the presence of traits associated with the lichen lifestyle. The genome of C. viridis provides a new resource for exploring the evolution of the lichen symbiosis, and how symbiotic lifestyles shaped evolution in green algae.
Coccoid microalgae of the genus Trebouxia Puymaly are by far the most prevalent among the various species involved in lichen symbioses. However, their taxonomic knowledge is rather scarce compared to that of lichenized fungi. In the present work, a taxonomic study integrating diverse techniques (phylogenetics, light, confocal and transmission electron microscopies) is carried out to describe Trebouxia maresiae Garrido-Benavent, Chiva & Barreno, sp. nov. This species widely associates with the red-listed lichenized fungus Seirophora villosa (Ach.) Frdn but also with species of the genus Ramalina Ach., both occurring in coastal environments in the western Mediterranean and the Cape Verdean islands. This microalga is circumscribed to Trebouxia clade A and is closely related to T. decolorans Ahmadjian. It is characterized by the cell size being up to 15 m in diam., the crenulate chloroplasts, and the structure of pyrenoids, which in cultured cells fits well with the crenulata-type, with long branched tubules meandering through the pyrenoid matrix, whereas in the lichenized state it acquires a hybrid structure (maresiae-type), characterized by the periphery of the pyrenoid being rather gigantea-type, with thylakoid membranes forming short, branched tubules. With the present work, the taxonomy of the genus Trebouxia moves a step forward towards more accurately characterizing species in lichen microalgae which is a prerequisite for future, more complex studies on speciation, co-evolution and selectivity. Trebouxia maresiae sp. nov. (Trebouxiophyceae, Chlorophyta), une espce nouvelle de microalgue lichnise prsente dans les environnements ctiers. Les microalgues cocodes du genre Trebouxia Puymaly sont les plus rpandues parmi les diffrentes espces associes aux symbioses lichniques. Cependant, la connaissance taxonomique de ces espces est plutt lacunaire par rapport celle des champignons lichniss. Dans ce travail, une tude taxonomique intgrant diverses techniques (phylogntique, microscopies optique, confocale et lectronique transmission) a t ralise pour dcrire Trebouxia maresiae Garrido-Benavent, Chiva & Barreno, sp. nov. Cette espce s'associe principalement au champignon lichnis Seirophora villosa (Ach.) Frdn, figurant sur la liste rouge, mais aussi avec des espces du genre Ramalina Ach., tous deux prsents dans les environnements ctiers de la Mditerrane occidentale et des les du Cap-Vert. Cette microalgue est circonscrite au clade A de Trebouxia, et dmontre une relation troite avec T. decolorans Ahmadjian. Elle se caractrise par la taille de ses cellules (jusqu' 15 m de diamtre), ses chloroplastes crnels et la structure de ses pyrnodes qui, dans les cellules cultives, correspond bien au crenulata-type, avec de longs tubules ramifis serpentant dans la matrice du pyrnode, alors qu' l'tat lichnis, elle adopte une structure hybride (maresiae-type), caractrise par la priphrie du pyrnode tant plutt du gigantea-type, avec les membranes des thylacodes formant des tubules courts et ramifis. Cette contribution la taxonomie du genre Trebouxia permet de faire avancer l'valuation plus prcise de la dlimitation des espces chez les microalgues lichniques, ce qui est une condition pralable des tudes futures plus complexes sur la spciation, la co-volution et la valeur slective.
