INFERRING TAXONOMIC POSITIONS AND TESTING GENUS LEVEL ASSIGNMENTS IN COCCOID GREEN LICHEN ALGAE: A PHYLOGENETIC ANALYSIS OF 18S RIBOSOMAL RNA SEQUENCES FROM DICTYOCHLOROPSIS RETICULATA AND FROM MEMBERS OF THE GENUS MYRMECIA (CHLOROPHYTA, TREBOUXIOPHYCEAE CL. NOV.)1

Complete nuclear-encoded small-subunit ribosomal RNA (18S rRNA) coding sequences were determined for the coccoid green algae Dictyochloropsis reticulata (Tschermak-Woess) Tschermak-Woess , Myrmecia astigmatica Vinatzer, and M. bisecta Reisigl, to investigate the taxonomic position of Dictyochloropsis Geitler and of the genus Myrmecia Printz. Phylogenies inferred from these data revealed a sister-group relationship between D. reticulata and certain coccoid green algae that lack motile stages (autosporic coccoids) within the order Microthamniales. The monophyletic origin of the Microthamniales, including autosporic coccoids previously classified in the Chlorophyceae, is clearly resolved by the rRNA sequence data. This finding. shows the considerable taxonomic breadth of that order, whose taxonomic position has been unclear so far. A new class, Trebouxiophyceae, is proposed for this group of green algae. Phylogenetic inferences from the rRNA sequences show paraphyly of the genus Myrmecia. The 18S rRNA sequence data suggest that, among taxa that share similar vegetative cell morphologies, the zoospore characters resolve better the actual genus and species boundaries. Within identical zoospore types, the rRNA data allow further resolution of taxonomic relationships. On the basis of the.se findings, I propose that the genus Friedmannia Chantanachat ± Bold be merged into Myrmecia and that only those species be left in the genus Myrmecia that are identical in particular zoospore characters (i.e. those described in detail for M. israeliensis ( Chantanachat ± Bold) comb, nov.), namely M. astigmatica, M. biatorellae (Tschermak-Woess ± Ptesst) Petersen, and M. israeliensis. Myrmecia bisecta has to be excluded from Myrmecia; its taxonomic position within the Trebouxiophyceae is unclear .     

ULTRASTRUCTURE AND PHYLOGENETIC RELATIONSHIPS OF CHAETOPELTIDALES ORD. NOV. (CHLOROPHYTA,CHLOROPHYCEAE)1

Vegetative cells and zoospores of Hormotilopsis gelatinosa Trainor & Bold, H. tetravacuolaris Arce & Bold, Planophila terrestris Groover & Hofstetter, and Phyllogloea fimbriata (Korchikov) Silva were examined by transmission electron microscopy. All cells had pyrenoids traversed by cytoplasmic channels. Zoospores were quadriflagellate and had essentially cruciate flagellar apparatuses. Scales were present on free-swimming zoospores. These features are essentially identical to those of Chaetopeltis sp. and are dissimilar to those of other described green algae. The new order Chaetopeltidales is created to accommodate the genera Chaetopeltis, Hormotilopsis, Planophila sensu Groover & Hofstetter, Phyllogloea, Dicranochaete, and Schizochlamys, organisms previously scattered among the orders Tetrasporales, Chloro-coccales, Chlorosarcinales, and Chaetophorales. Members of the order are closely related to the ancestral chlorophycean flagellate genus Hafniomonas, may be ancestral with respect to other Chlorophyceae, and may also be closely related to the ulvophycean order Ulotrichales.     

Ribosomal RNA sequence comparisons demonstrate an evolutionary relationship betweenZygnematales and charophytes

The nuclear-encoded small subunit ribosomal RNA (rRNA) sequences were determined forGenicularia spirotaenia, Mesotaenium caldariorum, andStaurastrum spec. (Zygnematales) to elucidate the evolutionary position of these green algae. Results of neighbour-joining and maximum parsimony phylogenetic analyses support a monophyletic origin of theZygnematales within the evolutionary assemblage defined by theCharophyceae (sensuMattox & Stewart) and land plants. TheZygnematales/Charophyceae/land plants are evolutionarily distinct from the monophyletic lineage defined by theChlorodendrales, Pseudoscourfieldiales, and theMicrothamniales/Chlorophyceae. In memoriamRobert W. Hoshaw.     

PHYLOGENY OF THE ULVOPHYCEAE (CHLOROPHYTA): CLADISTIC ANALYSIS OF NUCLEAR-ENCODED rRNA SEQUENCE DATA1

Cladistic analysis of nuclear-encoded rRNA sequence data provided us with the basis for some new hypotheses of relationships within the green algal class Ulvophyceae. The orders Ulotrichales and Ulvales are separated from the clade formed by the remaining orders of siphonous and siphonocladous Ulvophyceae (Caulerpales, Siphonocladales /Cladophorales [S/C] complex, and the Dasycladales), by the Chlorophyceae and Pleurastrophyceae. Our results suggest that the Ulvophyceae is not a monophyletic group. Examination of inter- and intra-ordinal relationships within the siphonous and siphonocladous ulvophycean algae revealed that Cladophora, Chaetomorpha, Anadyomene, Microdictyon, Cladophoropsis and Dictyosphaeria form a clade. Thus the hypothesis, based on ultrastructural features, that the Siphonocladales and Cladophorales are closely related is supported. Also, the Caulerpales is a monophyletic group with two lineages; Caulerpa, Halimeda, and Udotea comprise one, and Bryopsis and Codium comprise the other. The Dasycladales (Cymopolia and Batophora) also forms a clade, but this clade is not inferred to be the sister group to the S/C complex as has been proposed. Instead, it is either the sister taxon to the Caulerpales or basal to the Caulerpales and S/C clade The Trentepohliales is also included at the base of the siphonous and siphonocladous ulvophycean clade. The Pleurastrophyceae, which, like the Ulvophyceae, posses a counter-clockwise arrangement of flagellar basal bodies, are more closely related to the Chlorophyceae than to the Ulvophyceae based on rRNA sequences. Thus, the arrangement of basal bodies does not diagnose a monophyletic group. Previously reported hypotheses of phylogenetic relationships of ulvophycean algae were tested. In each case, additional evolutionary steps were required to obtain the proposed relationships. Relationships of ulvophycean algae to other classes of green algae are discussed.     

PRASINOPHYTES FORM INDEPENDENT LINEAGES WITHIN THE CHLOROPHYTA: EVIDENCE FROM RIBOSOMAL RNA SEQUENCE COMPARISONS1

Complete nuclear-encoded small-subunit ribosomal RNA (rRNA) sequences were determined from Nephroselmis olivacea Stein, Pseudoscourfieldia marina (Throndsen) Manton, Scherffelia dubia (Perty) Pascher, and Tetraselmis striata Butcher (Chlorophyta) to investigate the evolutionary position of these scaly green flagellates. Results of neighbor-joining and maximum parsimony phylogenetic analyses demonstrate at least two independent prasinophyte lineages defined by N. olivacea/P. marina and S. dubia/T. striata, which together with the Chlorophyceae, Pleurastrophyceae, and Ulvophyceae form a monophyletic group. Within this assemblage, N. olivacea and P. marina represent an early-diverging lineage that is evolutionarily distinct from the later-diverging S. dubia/T. striata clade. The branch point of the S. dubia/T. striata clade precedes the near-simultaneous radiation of the Chlorophyceae, Ulvophyceae, and Microthamniales. Though interrelationships between these three latter groups of algae are not resolved, the phylogenetic analyses demonstrate that the Prasinophyceae (sensu Moestrup and Throndsen) and the Pleurastrophyceae (sensu Mattox and Stewart) are not monophyletic classes.     

Morphological and phylogenetic study of algal partners associated with the lichen-forming fungus <Emphasis Type="Italic">Tephromela atra</Emphasis> from the Mediterranean region

Recent DNA sequence analyses have revealed the diversity of algal partners in lichen symbioses. Although morphologically similar, different genetic lineages of photobionts are detected in wide geographic ranges of the same lichen fungal species. We studied the photobiont of the genus Trebouxia, which are known as partners of diverse lichen-forming fungal species in the Mediterranean region. We studied the phylogeny of these algae with a multilocus dataset including three loci: ITS, rbcL, and actin type I gene. The two lineages found, informally named Trebouxia sp. 1 and Trebouxia sp. 2, are related to Trebouxia arboricola/decolorans. The cultivation under axenic conditions succeeded only for one of them so far. We used light microscopy, confocal laser scanning microscopy and transmission electron microscopy for phenotypic characterisation. The ultrastructural characters currently used to describe species in the genus do not support the segregation of Trebouxia sp.1 from Trebouxia arboricola. The preferential presence in Mediterranean climates of these strains suggests eco-physiological adaptation. Despite their asexuality in long living lichen symbioses, coccoid algal lichen partners have apparently diversified genetically and physiologically.     

PHYLOGENETIC POSITION OF BOLBOCOLEON PILIFERUM (ULVOPHYCEAE,CHLOROPHYTA): EVIDENCE FROM REPRODUCTION,ZOOSPORE AND GAMETE ULTRASTRUCTURE,AND SMALL SUBUNIT RRNA GENE SEQUENCES1

Aspects of the reproduction of Bolbocoleon piliferum N. Pringsheim, a common, small, filamentous, endophytic marine green alga, were examined by LM and TEM. These observations were combined with phylogenetic analysis of nuclear‐encoded small subunit rRNA gene sequences to assess the phylogenetic position of B. piliferum. Quadriflagellate zoospores and planozygotes derived from fusion of isogametes yielded plants with identical morphology. Zoosporangia and gametangia divided by sequential cleavages. Plugs at the apices of zoosporangia and gametangia formed during development; tubes were found at zoosporangial and gametangial apices after swarmer release. Flagellar apparatuses of zoospores and gametes were similar to those of algae in the Ulvales (Ulvophyceae), except that terminal caps were entire rather than bilobed and rhizoplasts and “stacked” microtubular root configurations were absent. Structures associated with planozygotes were identical to those observed in other algae currently assigned to Ulotrichales and Ulvales. Molecular phylogenetic analyses placed B. piliferum within the Ulvophyceae, at the base of a clade that contains representatives of the families Ulvaceae, Ulvellaceae, and Kornmanniaceae. The results support an earlier hypothesis that B. piliferum constitutes a distinct lineage. Analyses including Kornmanniaceae recover monophyletic Ulotrichales and Ulvales, whereas analyses omitting the Kornmanniaceae indicate that Ulotrichales is paraphyletic. The structures associated with gamete fusion are conserved within Ulotrichales and Ulvales and perhaps more widely within Chlorophyta.     

OCCURRENCE OF GLYCOLATE DEHYDROGENASE AND GLYCOLATE OXIDASE IN SOME COCCOID,ZOOSPORE-PRODUCING GREEN ALGAE1

Twenty-seven species of coccoid, zoospore-producing green algae representing 16 genera in the Chlorococcales and Chlorosarcinales were assayed for glycolate oxidase or glycolate dehydrogenase. Only Planophila terrestris Groover & Bold and Fasciculochloris boldii Trainor, contained glycolate oxidase whereas the others contained glycolate dehydrogenase. Representative algae were grown under varying conditions and assayed to determine any effects on these glycolate enzymes. Although specific rates of enzyme activity often varied widely, the form of glycolate enzyme present was not affected.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

Relationship between carbohydrate movement and the symbiosis in lichens with green algae

Summary When isolated in pure culture, four genera of lichen algae were able to produce the polyol which is known to move from the alga to the fungus in lichens with these algae. This conclusion corrects earlier suggestions that the mobile polyol is only formed by the alga in the lichen thallus. Stichococcus produced sorbitol and it is therefore suggested that, in lichens with this alga, sorbitol moves between the symbionts. Hyalococcus and Stichococcus had a similar pattern of incorporation of H¹⁴CO ₃ ^- in the light, suggesting a close relationship between these algae which are only separated now on morphological grounds.The pattern of incorporation of H¹⁴CO ₃ ^- in the light into Cladonia cristatella and its alga (Trebouxia erici) in culture indicates that in the cultured algae more ¹⁴C was incorporated into ethanol insoluble substances and lipids and less into ribitol than in the lichen. The pattern in a joint culture of the alga and the fungus of C. cristatella was approximately intermediate between that of the lichen and the alga. However, only a small amount of ¹⁴C fixed by the alga reached the fungus in the joint culture, and it is therefore suggested that the presence of the fungus without morphological differentiation into a lichen thallus is not sufficient to promote the alga to release carbohydrate.     

Phylogenetic relationships of green algae assigned to the genus Planophila (Chlorophyta): evidence from 18S rDNA sequence data and ultrastructure

Phylogenetic analyses, based upon nuclear small-subunit ribosomal RNA gene sequences, of four ‘chlorosarcinoid’ species referred to Planophila Gerneck show that the genus is polyphyletic. The type species, P. laetevirens Gerneck, is closely related to species in the Ulotrichales, Ulvophyceae. The monotypic sarcinoid genus Pseudendocloniopsis is the closest relative of Planophila; the two genera represent the addition of a new morphological type to the Ulotrichales. Planophila microcystis (Dangeard) Kornmann & Sahling forms a clade at the base of the Ulvophyceae with Oltmannsiellopsis, and thus belongs to the Oltmannsiellopsidales. This result is also supported by the Oltmannsiellopsis-like ultrastructure of P. microcystis zoospores. Planophila sp. B from Antarctica, which has Trebouxia-like pyrenoid structure, is a trebouxiophyte closely related to Chlorella-like unicellular coccoids, Stichococcus bacillaris and Prasiola species. This is the first robustly supported molecular phylogenetic analysis that places Prasiola in the Trebouxiophyceae. As shown previously, P. terrestris Groover & Hofstetter belongs to the Chaetopeltidales, Chlorophyceae. Dangemannia gen. nov. (type species : D. microcystis (Dangeard) comb. nov.), Floydiella gen. nov. (type species : F. terrestris (Groover & Hofstetter) comb. nov.) and Pabia gen. nov. (type: P. signiensis sp. nov.) are proposed.     

Influence of long-term exposure to copper on the lichen photobiont <Emphasis Type="Italic">Trebouxia erici</Emphasis> and the free-living algae <Emphasis Type="Italic">Scenedesmus quadricauda</Emphasis>

In the present work, the long-term effect (14 days) of copper on the levels of intracellular and total copper accumulation, growth, assimilation pigment composition, chlorophyll a fluorescence, soluble protein content and oxidative status (production of hydrogen peroxide and superoxide) in two algal species (Scenedesmus quadricauda and Trebouxia erici) was assessed. Scenedesmus quadricauda is a free-living alga while Trebouxia erici is the photobiont of a lichen. The presence of copper negatively affected growth, assimilation pigments, chlorophyll a fluorescence, soluble protein content and oxidative status in both the algae. However, Scenedesmus was much more sensitive compared to Trebouxia.     

SSU rRNA GENE PHYLOGENY OF MORPHOSPECIES AFFILIATED TO THE BIOASSAY ALGA “SELENASTRUM CAPRICORNUTUM” RECOVERED THE POLYPHYLETIC ORIGIN OF CRESCENT‐SHAPED CHLOROPHYTA1

The generic concept of coccoid green algae exhibiting a crescent‐shaped morphotype is evaluated using SSU rRNA gene sequence analyses and light and electron microscopical observations. These common chlorophytes evolved polyphyletically in 10 different clades of the Chlorophyceae and three clades of the Trebouxiophyceae. Six clades are assigned to known genera of Selenastraceae: Kirchneriella, Nephrochlamys, Raphidocelis, Rhombocystis, Selenastrum, and Tetranephris. Four other clades, named following their present genus designation as Ankistrodesmus‐like I and II and Monoraphidium‐like I and II, require further investigation. One crescent‐shaped morphotype, which evolved within the Trebouxiophyceae, is designated as Neocystis mucosa sp. nov. The other two lineages containing trebouxiophycean algae with this morphotype are the Elliptochloris and the Watanabea clades. The taxonomic placement of the widely used bioassay strain “Selenastrum capricornutum” NIVA‐CHL 1 in the genus Raphidocelis (species name Raphidocelis subcapitata) is indicated by molecular data.     

TWO SNOW SPECIES OF THE QUADRIFLAGELLATE GREEN ALGA CHLAINOMONAS (CHLOROPHYTA,VOLVOCALES): ULTRASTRUCTURE AND PHYLOGENETIC POSITION WITHIN THE CHLOROMONAS CLADE1

The quadriflagellate snow alga Chlainomonas Christen, distributed in New Zealand and North America, has several unusual structural attributes. A process assumed to be cytokinesis involves extrusion of protoplasm from the parent through a narrow canal, C. kolii (J. T. Hardy et Curl) Hoham produces a net‐like outer envelope rather than a cell wall, and the flagellar basal apparatus of Chlainomonas consists of two semi‐independent pairs of basal bodies. Structural connections between basal body pairs appear minimal, but a connecting system different from that observed in other genera exists within each pair. Phylogenetic analysis using rbcL sequences places Chlainomonas in the Chloromonas clade, other known members of which are all biflagellate. Chlainomonas is split into two robust lineages, with New Zealand collections sharing an origin with northern North American collections. Although the quadriflagellate condition is regarded as ancestral in the Chlorophyceae, we speculate—based on ultrastructural and molecular data presented here—that Chlainomonas represents a derived form that has arisen from fusion of two ancestral biflagellate cells. Other explanations (for example, that Chlainomonas represents a diploid form of a biflagellate species) are remotely possible but are presently at odds with extensive observations of field material. Improvements in techniques for experimental manipulation of these sensitive cryophiles will be required to fully characterize their structure and progress our understanding of their biology.     

Comparative ultrastructure of pyrenoids inTrebouxia (Microthamniales,Chlorophyta)

Pyrenoid ultrastructure has been investigated from cultures of all 26 species ofTrebouxia with the aim of establishing pyrenoids as a taxonomic character. Different arrangements and forms of thylakoid lamellae within the pyrenoid matrix allow eight pyrenoid types to be distinguished. Each type is characteristic of a group of species. Thegigantea- andimpressa-type are similar, differing only in the form of the tubules: short, branched tubules mark thegigantea-type; ± long and straight invaginations theimpressa-type. Thearboricola-type is characterized by meandering pyrenoid membranes developing from lamellae parallel with each other in young autospores. Pyrenoids of thegelatinosa-type are traversed by thin parallel-arranged tubules. Few thylakoids with a curved profile are typical of theirregularis-type. Thecorticola-type is different from all others in having a distinct starch sheath closely connected with the pyrenoid matrix and no pyrenoglobuli being associated with the pyrenoid membranes. No true pyrenoids have been found inT. magna andT. erici. Within the chloroplast, they have indistinct areas with pyrenoglobuli, but without differentiated thylakoids. Pyrenoid morphology is stable in culture on different media as well as in phycobionts within lichen thalli. Comparing the pyrenoid of a lichenizedTrebouxia with that from cultured species, the identification of the phycobiont within the lichen thallus is possible, without the need of culturing the algae. This has been shown in species ofParmelia andHypogymnia. New aspects for the taxonomy and systematics ofTrebouxia are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

EVIDENCE THAT THE NUCLEOMORPHS OF CHLORARACHNION REPTANS (CHLORARACHNIOPHYCEAE) ARE VESTIGIAL NUCLEI: MORPHOLOGY,DIVISION AND DNA-DAPI FLUORESCENCE1

Chlorarachnion reptans Geitler shows affinities to both the Chlorophyceae and the chloroplast endoplasmic reticulum-containing chromophyte algae in possessing chlorophyll b and chloroplasts which are limited by four membranes, respectively. In the periplastidal compartment surrounding each of the four to eight chloroplasts of a C. reptans cell are putative eukaryotic-sized ribosomes, scattered tubules and vesicles, and a small double-membrane-limited nucleus-like organelle named the nucleomorph. The nucleomorphs display 4′-6-diamidino-2-phenylindole (DAPI)fluorescence which is sensitive to DNase digestion, but not to treatment with RNase. The nucleomorphs also contain a fibrillogranular body which resembles a nucleolus. Nucleomorph division occurs by the sequential infolding of the inner and outer envelope membranes and subsequent constriction in two, with no involvement of microtubules. In all these characteristics, the nucleomorphs of C. reptans are similar to the cryptomonad nucleomorph which has been hypothesized to be the vestigial nucleus of an ancestral red alga which gave rise to the chloroplasts of the Cryptophyceae. The presence of chlorophyll b and the contents and morphology of C. reptans chloroplast compartments suggest a green algal origin for the chloroplasts of these cells. The discovery of a second organism with a DNA-containing, nucleus-like organelle in its chloroplast compartment lends strong support to the hypothesis that the chloroplasts of many algae have evolved from eukaryotic endosymbionts.     

SMITHSONIELLA GEN. NOV., A POSSIBLE EVOLUTIONARY LINK BETWEEN THE MULTICELLULAR AND SIPHONOUS HABITS IN THE ULVOPHYCEAE,CHLOROPHYTA

A low relief, green turf-forming alga of a heterotrichous habit was discovered in the coral reef microcosm, Museum of Natural History, Smithsonian Institution. Erect filaments bore lateral, specialized sporangia and together with basal filaments possessed septal plugs between adjacent cells, grossly similar to the “pit connections” of red algae. Data are presented which: 1) establish the identity of our plant with a plant recently described as Pilinia earleae Gallagher et Humm from the Florida Gulf coast; 2) support our establishment of the new genus Smithsoniella and our transfer of P. earleae to this new taxon. Additional data on pigmentation and cytology are related to the fine structure of other selected green algae to develop and test three hypotheses, viz. Smithsoniella earleae represents either: 1) a symbiotic association between a green and a red alga; 2) an alga which belongs to either the Ulotrichales, Chaetophorales or the Chroolepidales; or 3) an alga representing an evolutionary link between filamentous forms of the Ulvophyceae and members of the coenocytic siphonalean complex (e.g., Codiales or Caulerpales) of the Chlorophyta. Data refute hypotheses 1 and 2 but do lend support to the third hypothesis.     

基于核及叶绿体基因序列探讨中国绿水螅共生单细胞绿藻系统发生地位

研究对中国绿水螅共生绿藻的核18S rRNA基因全长序列及其叶绿体9个基因(atpA、chlB、chlN、petA、psaB、psbA、psbC、psbD及rbcL)片段序列进行了克隆和测序, 并基于18S rRNA基因序列及叶绿体9个基因序列的整合数据分别通过最大似然法(Maximum-likelihood)和贝叶斯分析(Bayesian inference)对中国绿水螅(Hydra sinensis)共生单细胞绿藻的系统发生地位进行了探讨。系统发生表明: (1)中国绿水螅共生绿藻属于共球藻纲(Trebouxiophyceae)小球藻目(Chlorellales), 但不属于其中的小球藻属(Chlorella); (2)来源于草履虫、水螅、地衣及银杏的共生绿藻均在共球藻纲支系, 而来源于蛙类和蝾螈的共生绿藻属于绿藻纲(Chlorophyceae)支系。无论在共球藻纲支系还是在绿藻纲支系, 不同来源的共生藻并没有排他性地聚为单系群而在系统树中与其他自由生活的绿藻混杂排列, 来自不同宿主的共生绿藻没有共同起源。     

Differenze Ultrastrutturali Di Alcune Specie Di Trebouxia Poste in Condizioni Di Illuminazione Differenti

Abstract

Ultrastructural changes in some species of « Trebouxia » under different light conditions. — Some species of the phycobiont alga Trebouxia (Tr. decolorans and Tr. albulescens), both isolated and grown on synthetic medium and still in the lichen, were examined in order to study the effect of light on the plastid ultrastructures. The species isolated from Buellia punctata and Xanthoria parietina were very sensitive to light condition and lost their chlorophyll content quickly. Striking ultrastructural changes were found in the algae grown under small light intensities and those which become achloric owing to strong light. In the latter, modifications of the Iamellar System were observed. The disappearance of Chlorophyll pigments was followed by a reduced electron density of the whole Iamellar system, as if were lacking the Iipidic compounds which are usually present and absorb fixators and dyers, thus allowing a good view. On the contrary, normal light conditions did not affect cultures of Trebouxia humicola, a free living alga. In the chloroplasts of the phycobiont species, unlike in the free living alga, grana were very close and sometimes formed very thick masses towards the edge of the chloroplast. It could not be ascertained whether such changes corresponded to a different composition of the lipoproteic compounds of the lamellar system.

Xanthoria parietina could grow in very lighted environments with no damage of the algae present in its thallus. The lichen thalluses, under different light conditions, showed very different colourings: the overlighted ones were rusty-red and the shadowed ones deep green. The chlorophyll content of the lichen thalluses with various shades (table 1) were very similar. The ultrastructural changes induced by strong light intensities in the phycobiont algae, kept in the lichen, were very small in respect of those observed in the same algae isolated and grown on synthetic medium and concerned the Iamellar system and the pyrenoid, above all. The rusty-red lichen showed a great number of stromatic lamellae, often with a parallel trend, so as to simulate a Iamellar system not organized in grana and often presented groups of lamellae concentrically arranged. In the pyrenoid of the algae from rusty-red thalluses, compared with the green ones, a much greater number of electron dense masses was observed, which are very thick and occupy the whole stromatic portion of the pyrenoid. But the Chlorophyll content did not decrease. Unlike the results of PEVELING, we noted that the electron dense masses (cited by the Author as « osmiophilic plastoglobules) were visible even after fixation with permanganate; the different numbers of these globules might depend on environmental factors. The phycobiont alga, when in the lichen thallus, could perhaps support strong light intensities, because pigments or compounds formed with the mycobiont or by it alone prevented the photooxidation of chlorophyll. Hypothetically a relationship might exist between the sensitivity of the phycobiont algae to light intensities and the content in antraquinonic pigments in the lichen thallus. But also using filters with absorption maxima similar to those of these pigments, the « in vitro » cultures of the phycobiont algae became achloric in the same time as the control ones.

Some Authors had found in Trebouxia humicola a different relationship between Chlorophyll pigments and carotinoids from that observed in the phycobiont species and had ascribed to it the greater resistence to strong light of the free living alga. Pigments or other substances present in the mycobiont can have a protective action on the Chlorophyll content and on the ultrastructures. In the phycobiont algae the resistence to strong light might be explained by an exchange of compounds between mycobiont and phycobiont, ending with the structural changes of the pyrenoid.