Ecophysiological changes and spore formation: two strategies in response to low-temperature and high-light stress in Klebsormidium cf. flaccidum (Klebsormidiophyceae, Streptophyta)1.

Members of the cosmopolitan streptophycean genus Klebsormidium live in various habitats, including sand dunes and polar/alpine environments. To survive in these harsh conditions they must possess an array of adaptive physiological and structural mechanisms, for example, to deal with chilling and photochilling stresses. Since these mechanisms have not been studied in detail, the objectives of this study were (i) to determine the physiological and biochemical responses of Klebsormidium cf. flaccidum (K. cf. flaccidum) to chilling (low temperature [LT]) and photochilling (LT in combination with high light [HL]) stresses; and (ii) to understand the cross-link between biochemical parameters and cellular ultrastructural changes. The results indicated that 5°C is a temperature threshold (i.e., at 5°C) but not at higher temperatures, physiological changes were observed (Fv /Fm and ETR decreased and energy-partitioning distribution changed, with an increase in Y[NPQ] under LT and an increase in Y[NO] under HL-LT). Also, pigment contents changed significantly, with increased concentrations of photoprotective pigments such as antheraxanthin, zeaxanthin, and total carotenes. All of these responses occurred under LT and, to a greater extent, under LT-HL, indicating that the two stresses (temperature and light) are additive. The cold treatment applied here induced the formation of spores under both LL and HL. The degree of photoinhibition was higher in spores than in vegetative cells, indicating that spores are less susceptible to photodamage. This study demonstrated a broad acclimation potential in different developmental stages of K. cf. flaccidum, which helps to explain the ecological success of this genus.

Unraveling the Photoprotective Response of Lichenized and Free-Living Green Algae (Trebouxiophyceae, Chlorophyta) to Photochilling Stress.

Lichens and free-living terrestrial algae are widespread across many habitats and develop successfully in ecosystems where a cold winter limits survival. With the goal of comparing photoprotective responses in free-living and lichenized algae, the physiological responses to chilling and photochilling conditions were studied in three lichens and their isolated algal photobionts together as well as in a fourth free-living algal species. We specifically addressed the following questions: (i) Are there general patterns of acclimation in green algae under chilling and photochilling stresses? (ii) Do free-living algae exhibit a similar pattern of responses as their lichenized counterparts? (iii) Are these responses influenced by the selection pressure of environmental conditions or by the phylogenetic position of each species? To answer these questions, photosynthetic fluorescence measurements as well as pigment and low molecular weight carbohydrate pool analyses were performed under controlled laboratory conditions. In general, photochemical efficiency in all free-living algae decreased with increasing duration of the stress, while the majority of lichens maintained an unchanged photochemical activity. Nevertheless, these patterns cannot be generalized because the alga Trebouxia arboricola and the lichen Ramalina pollinaria (associated with Trebouxia photobionts) both showed a similar decrease in photochemical efficiency. In contrast, in the couple Elliptochloris bilobata-Baeomyces rufus, only the algal partner exhibited a broad physiological performance under stress. This study also highlights the importance of the xanthophyll cycle in response to the studied lichens and algae to photochilling stress, while the accumulation of sugars was not related to cold acclimation, except in the alga E. bilobata. The differences in response patterns detected among species can be mainly explained by their geographic origin, although the phylogenetic position should also be considered, especially in some species.

Species concept and nomenclatural changes within the genera Elliptochloris and Pseudochlorella (Trebouxiophyceae) based on an integrative approach.

The genera Elliptochloris and Pseudochlorella were erected for Chlorella-like green algae producing two types of autospores and cell packages, respectively. Both genera are widely distributed in different soil habitats, either as free living or as photobionts of lichens. The species of these genera are often difficult to identify because of the high phenotypic plasticity and occasional lack of characteristic features. The taxonomic and nomenclatural status of these species, therefore, remains unclear. In this study, 34 strains were investigated using an integrative approach. Phylogenetic analyses demonstrated that the isolates belong to two independent lineages of the Trebouxiophyceae (Elliptochloris and Prasiola clades) and confirmed that the genera are not closely related. The comparison of morphology, molecular phylogeny, and analyses of secondary structures of SSU and ITS rDNA sequences revealed that all of the strains belong to three genera: Elliptochloris, Pseudochlorella, and Edaphochlorella. As a consequence of the taxonomic revisions, we propose two new combinations (Elliptochloris antarctica and Pseudochlorella signiensis) and validate Elliptochloris reniformis, which is invalidly described according to the International Code for Nomenclature (ICN), by designating a holotype. To reflect the high phenotypic plasticity of P. signiensis, two new varieties were described: P. signiensis var. magna and P. signiensis var. communis. Chlorella mirabilis was not closely related to any of these genera and was, therefore, transferred to the new genus Edaphochlorella. All of the taxonomic changes were highly supported by all phylogenetic analyses and were confirmed by the ITS-2 Barcodes using the ITS-2/CBC approach.

Mixotrophy in the terrestrial green alga Apatococcus lobatus (Trebouxiophyceae, Chlorophyta).

The green microalga Apatococcus lobatus is widely distributed in terrestrial habitats throughout many climatic zones. It dominates green biofilms on natural and artificial substrata in temperate latitudes and is regarded as a key genus of obligate terrestrial consortia. Until now, its isolation, cultivation and application as a terrestrial model organism has been hampered by slow growth rates and low growth capacities. A mixotrophic culturing approach clearly enhanced the accumulation of biomass, thereby permitting the future application of A. lobatus in different types of bio-assays necessary for material and biofilm research. The ability of A. lobatus to grow mixotrophically is assumed as a competitive advantage in terrestrial habitats.

Evaluating the Species Boundaries of Green Microalgae (Coccomyxa, Trebouxiophyceae, Chlorophyta) Using Integrative Taxonomy and DNA Barcoding with Further Implications for the Species Identification in Environmental Samples.

Integrative taxonomy is an approach for defining species and genera by taking phylogenetic, morphological, physiological, and ecological data into account. This approach is appropriate for microalgae, where morphological convergence and high levels of morphological plasticity complicate the application of the traditional classification. Although DNA barcode markers are well-established for animals, fungi, and higher plants, there is an ongoing discussion about suitable markers for microalgae and protists because these organisms are genetically more diverse compared to the former groups. To solve these problems, we assess the usage of a polyphasic approach combining phenotypic and genetic parameters for species and generic characterization. The application of barcode markers for database queries further allows conclusions about the 'coverage' of culture-based approaches in biodiversity studies and integrates additional aspects into modern taxonomic concepts. Although the culture-dependent approach revealed three new lineages, which are described as new species in this paper, the culture-independent analyses discovered additional putative new species. We evaluated three barcode markers (V4, V9 and ITS-2 regions, nuclear ribosomal operon) and studied the morphological and physiological plasticity of Coccomyxa, which became a model organism because its whole genome sequence has been published. In addition, several biotechnological patents have been registered for Coccomyxa. Coccomyxa representatives are distributed worldwide, are free-living or in symbioses, and colonize terrestrial and aquatic habitats. We investigated more than 40 strains and reviewed the biodiversity and biogeographical distribution of Coccomyxa species using DNA barcoding. The genus Coccomyxa formed a monophyletic group within the Trebouxiophyceae separated into seven independent phylogenetic lineages representing species. Summarizing, the combination of different characteristics in an integrative approach helps to evaluate environmental data and clearly identifies microalgae at generic and species levels.

POLYOL PATTERNS IN BIOFILM-FORMING AEROTERRESTRIAL GREEN ALGAE (TREBOUXIOPHYCEAE, CHLOROPHYTA)1.

The distribution of polyols was examined for the first time in 34 green algal strains from four different clades belonging to the class Trebouxiophyceae, which dominate aeroterrestrial biofilms of many regions. Sorbitol was detected in representatives of the Prasiola clade, while ribitol was present in the Elliptochloris and Watanabea clades. Apatococcus lobatus (Chodat) J. B. Petersen contained erythritol in addition to ribitol. Polyols are considered as effective stress metabolites exerting multiple protective functions in metabolism and hence mainly occur in organisms colonizing extreme environments. In contrast, members of the Chlorella clade, which mainly occur in aquatic habitats, did not contain polyols. Thus, the constitutive presence of specific polyols facilitates a differentiation between species of the Prasiola clade from the Elliptochloris and Watanabea clades, respectively, and further allows differentiation of morphologically converging taxa.

Physiological and biochemical responses of green microalgae from different habitats to osmotic and matric stress.

Growth of five aeroterrestrial green algal strains (Trebouxiophyceae) in response to changing water availabilities-caused by osmotic (ionic) and matric (desiccation) stresses-was investigated in comparison with a freshwater and a marine strain. All investigated algae displayed good growth under brackish conditions while four out of the five aeroterrestrial strains even grew well under full marine conditions (28-40 psu). The comparison between growth responses in liquid medium, on solid agarose, and on glass fiber filters at 100% air humidity indicated a broad growth tolerance of aeroterrestrial algae towards diminished water availability. While two aeroterrestrial strains even grew better on solid medium which mimics natural biofilm conditions, the aquatic strains showed significant growth inhibition under matric stress. Except Stichococcus sp., which contained the C6-polyol sorbitol, all other aeroterrestrial green algae investigated synthesized and accumulated the C5-polyol ribitol in response to osmotic stress. Using (13)C NMR spectroscopy and HPLC, it could be verified that ribitol functions as an osmotically regulated organic solute. This is the first proof of ribitol in free-living aeroterrestrial green algae. The biochemical capability to synthesize polyols under environmental stress conditions seems to support algal life outside aquatic habitats.