Photobionts of the Physciaceae and the genus Trebouxia

Helms, G. & Friedl, T.
Experimentelle Phykologie und Sammlung für Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany

The genetic diversity of Physciacean photobionts, based on nrITS sequencing, has been investigated. A much larger genetic diversity was observed than had been known from cultured strains. This diversity was structured utilizing phylogenetic analyses, comparisons with authentic strains, as well as mycobiont selection behavior. Three systematic categories were employed to represent Trebouxia taxa. ITS sequences that differed in less than 2% of their nucleotide positions and were monophyletic in phylogenetic analyses (Neighbor-Joining, Jukes-Cantor model of evolution) were assigned to particular ITS-variants. Monophyletic groups of ITS-variants with p-distances usually less than 6 % were united into subclades. Subclades are supposed to refer to the taxonomic level of species. Approximately twice as many subclades were differentiated in the Physciaceae than were represented by authentic strains. Almost all Trebouxia strains known from other lichen families were also found in the Physciaceae. However, representatives of Clade S were found only exceptionally. According to their relatedness, subclades were summarized in clades following the clade concept of Friedl et al. (2000). This clade concept divides the genus Trebouxia s. str. into four major clades that receive significant support for monophyly in phylogenetic analyses except for clade S. Compiling the data that were published before this study, it seems evident that Trebouxia s.l. is a non-natural taxon which comprises two well separated lineages, Asterochloris and Trebouxia s.str., which need to be considered as two distinct genera. Both lineages vary greatly in their phylogenetic diversity. The diversity of ITS sequences of all known Asterochloris species corresponds to the diversity of just one subclade of Trebouxia s.str.


Photobiont specifity of cyanobacterial lichens – evidence from epiphytic communities

Myllys, L. (1), Stenroos, S. (1) & Kuusinen, M. (2)
(1) Herbarium, Department of Biology, FIN-20014 University of Turku, Finland; (2) Ministry of the Environment, P. O. Box 35 FIN-00023 Government, Finland

Samples of cyanobacterial epiphytic lichens of the genera Lobaria, Nephroma, Peltigera and Parmeliella were collected in a southern boreal old-growth forest area from (1) the same host tree, and (2) adjacent trees in the same stand. The photobionts of these samples were compared to free and symbiotic Nostoc obtained from other habitats and geographic regions to examine the photobiont diversity and specifity at different spatial scales. Our preliminary results, based on the partial small subunit of the ribosomal DNA (16S rDNA) and the rbcLX gene complex, show no tendency towards photobiont sharing, at least between distantly related species. Even thalli of different species occurring in close contact on the same phorophyte have their own, taxon-specific photobiont.


Patterns of specificity in cyanobacterial lichen symbioses

O’Brien, H., Miadlikowska, J. & Lutzoni, F.
Department of Biology, Duke University, Durham NC 27708 USA

Molecular phylogenetic studies of cyanobacterial photobionts of the genus Nostoc have reached contradictory conclusions about the extent of specificity in the cyanolichen symbiosis. Much of this confusion is due to differences in the taxonomic and geographical scales of sampling, as well as peculiarities of the genetic markers used. We have sampled multiple individuals of diverse cyanolichen species in the Peltigerales from nested spatial scales ranging from one meter sample plots to continental scales and sequenced commonly used cyanobacterial markers (16S rDNA, trnL), as well as three protein coding loci (rbcLX, rpoC2, nifV1). We have analyzed these data in the context of a broader phylogeny of free-living and symbiotic Nostoc, as well as other cyanobacterial genera to determine if the photobiont strains form a distinct lineage within the heterocystous cyanobacteria. We find that the degree of photobiont specificity varies on a species-by-species basis: some species appear to be specialized on a single photobiont strain over continental scales while many others share the same pool of photobiont strains at a single locality and lichenize with diverse Nostoc strains across their range. These results highlight the danger about making general statements about photobiont specificity and the evolutionary forces that have shaped it without first sampling a variety of different species at ecologically relevant spatial scales.


tRNALeu (UAA) intron morphotypes among lichen-forming Nostoc strains

Rikkinen, J.
Department of Biological and Environmental Sciences, University of Helsinki

Diversity and specificity patterns among symbiotic Nostoc strains have been studied by using tRNALeu (UAA) intron sequences as a genetic marker. The stable secondary and tertiary structure of trnL introns strictly limits possibilities for random mutations. The low number of informative characters, in turn, does not provide enough variation for hierarchical analysis and this restricts phylogenetic group formation. Here, relations between different trnL intron sequences were analyzed with multivariate methods. The sequences were first incorporated into a large alignment spanning different groups of filamentous cyanobacteria and including all Nostoc trnL intron sequences in GenBank. After reductions the data matrix was analyzed using ordination methods (non-metric multidimensional scaling, Bray-Curtis ordination) and the statistical significance of differences between sequence groups was estimated using Monte Carlo methods (MRPP). In the resulting ordinations most Nostocalean intron sequences grouped away from those of non-Nostocalean cyanobacteria. Furthermore, most Nostoc sequences were well separated from those of other Nostocalean genera. Three main sequence morphotypes, the Muscorum-, Commune- and Punctiformis-type, were delimited from the main cluster of Nostoc intron sequences. All sequences so far amplified from lichens have belonged to the latter two groups. The intron morphotypes seemed to broadly correspond with classical Nostoc species recognized on the basis of morphology and life-history traits. Furthermore, they were not in obvious conflict with phylogenetic classifications based on the 16S rRNA gene and/or the conserved parts of the trnL intron. Thus, intron morphotypes could be used for distinguishing ecological, geographic and/or taxonomic entities within the genus Nostoc, and aid in the rapid placement of newly acquired trnL intron sequences into a meaningful context.


Symbiont transformation in the unique phaeolichen formed by Verrucaria tavaresiae and its brown algal phycobiont Petroderma maculiforme

Sanders, W. B. (1, 2), Moe, R. L. (2) & Ascaso, C. (1)
(1) Centro de Ciencias Medioambientales, CSIC, Calle Serrano 115 bis, 28006 Madrid, Spain; (2) University Herbarium, University of California, Berkeley, USA

The recently described intertidal fungus Verrucaria tavaresiae Moe is the only species known to form a lichen association with a brown alga. Other symbioses between ascomycete fungi and brown algae have been considered mycophycobioses rather than lichens, because in those associations the fungus merely invades the tissue of the algal macrophyte without forming a distinctive thallus. A structural study using light and transmission electron microscopy was carried out on symbiotic thalli of V. tavaresiae and on its free-living phycobiont Petroderma maculiforme, both collected from San Francisco Bay, California (USA). Free-living Petroderma forms a crustose thallus of radiating, closely-appressed horizontal filaments that give rise to erect, adherent filaments often bearing terminal zooangia. In the lichen thallus, the phycobiont forms separate, anticlinally-oriented filaments that are interspersed within a cellular fungal tissue. The lichenized algal filaments appear to grow and branch into the thallus interior, and were not seen to produce zooangia. Their cell walls were thinner and storage bodies were much reduced compared to free-living Petroderma thalli. The plastid had a large pyrenoid that was either enfolded by plastid lobes or bulged laterally from the surface; this pyrenoid was penetrated by a system of tubules arising from invaginations of the plastid membrane system. A secondary, exserted pyrenoid with a narrow base and lacking tubules was also observed in some cells of lichenized Petroderma, but not in the free-living form. Verrucaria tavaresiae formed a previously unreported type of intraparietal "haustorium" that spread laterally between phycobiont wall layers. The lower apices of the algal filaments grew intrusively into the fungal tissue, forming reciprocal penetrations into the fungal wall material. These remarkable interpenetrations between symbionts may serve to integrate their divergent growth properties into a coherent tissue.


High diversity of sub-alpine aquatic lichens – is it determined by the submersion resistance of lichen photobionts?

Thüs, H. (1), Helms, G. (2), Friedl, T. (2) , Büdel, B. (1)
(1) Allgemeine Botanik, Universität Kaiserslautern, Germany; (2) Albrecht von Haller Institut, Universität Göttingen, Germany

Lichen photobionts from aquatic and semi-aquatic micro- sites of Central European creeks from low mountain ranges to sub alpine areas were identified by means of morphological (all taxa) and molecular characters (representative strains of Trebouxia s.l.). Nine different photobiont taxa were isolated from lichens of sub-alpine creeks. All algae occurred lichenized in the splash-zone as well as in habitats that were submerged for at least several months a year. At lower altitudes most of these photobionts were present in the splash-zone but only two of them, Dilabifilum sp. and Heterococcus caespitosus grew lichenized in long-term submerged micro-sites. In freshwater habitats Dilabifilum sp. and Heterococcus caespitosus were exclusively associated with a small group of Verrucaria species, most of them being adapted to aquatic and amphibic habitats. We conclude that at high altitudes the submersion tolerance of lichenized algae is generally higher than at low elevations of comparable latitudes. Dilabifilum sp. and Heterococcus caespitosus appeared as the only suitable photobionts for lichen symbiosis in submerged habitats at these low altitudes. The only genus of lichen forming fungi compatible with these two genera seemed to be members of the genus Verrucaria. The exceedingly poor lichen diversity at submerged micro-sites at low elevations might be in part a result of this specificity. We suggest that constantly low temperatures in upland watercourses and a resulting high gas holding capacity of the water allows a large number of algal taxa to dwell submersed, while at low altitudes with higher water temperatures in summer, especially CO2-availabilty may become limiting for submersed lichens. We suggest that Dilabifilum sp. and Heterococcus caespitosus, both possess highly effective pathways to concentrate CO2 which allow them to live as lichen photobionts even in waterbodies with a rather high (summer-) temperature and reduced gas holding capacity.


Geographic variation in photobiont specificity points to ecological, not evolutionary, specialization in Cladonia

Yahr, R. (1, 2), Vilgalys, R. (1), DePriest, P. T. (2)
(1) Duke University, Department of Biology, Box 90338, Durham, NC 27708 USA; (2) Smithsonian Institution, National Museum of Natural History, Washington, D.C. 20560 USA

In lichen symbioses, a fungus that is a photobiont specialist may be a better competitor than a generalist in a particular ecological setting. However, few lichen associations have been examined in detail across communities, and the ecological and evolutionary mechanisms dictating associations are generally poorly known. We intensively studied a suite of lichens on two spatial scales. Eight Cladonia species were sampled from five Florida scrub sites, for a total of more than 200 samples. One of these, C. subtenuis, was sampled in several communities across eastern North America to investigate geographic and ecological variation in photobiont specificity. We used ITS sequences of both Cladonia and Asterochloris for phylogenetic and population genetic analyses and identified three major photobiont lineages or clades. At the community level, all Florida scrub sites with the same fungal species contained a statistically equivalent set of potential Cladonia photobionts, i.e. photobionts from each of the three clades were available in each site. At this same spatial scale, however, most fungal species including C. subtenuis were only associated with a single Asterochloris clade. On a regional scale, C. subtenuis photobiont specificity was lower, including three additional algal clades. At all scales studied, fungi associated in unequal frequencies with compatible genotypes or clades. These associations were independent of fungal phylogeny, indicating strong fungal selectivity. At the larger geographic scale, these frequencies of photobiont association are correlated with latitude and physiographic region. Therefore, lichen associations may be products of ecological specialization of fungi for photobionts, perhaps linked with fitness benefits of preferential partnership across sites.

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