Phylogenetic signal of photobiont switches in the lichen genus <Emphasis Type="Italic">Pseudocyphellaria</Emphasis> s. l. follows a Brownian motion model

Lichen symbioses are defined as a symbiotic relationship between a mycobiont (generally an ascomycete) and one or more photobionts (green algae or/and cyanobacteria). It was proposed that cephalodia emancipation is an evolutionary driver for photobiont switch from chlorophyte to cyanobacteria. In this study we want to test the monophyly of cyanolichens and to measure the phylogenetic signal of the symbiotic relationship between cyanobacteria and a mycobiont partner in the lichen genus Pseudocyphellaria. This genus includes some species that have a chlorophyte as primary photobiont (and Nostoc in internal cephalodia), while others have only cyanobacteria. In a phylogenetic framework we measure the phylogenetic signal (or phylogenetic dispersion) as well as mapped photobiont switches performing stochastic character mapping. Results show that having cyanobacteria as main photobiont has a strong phylogenetic signal that follows a Brownian motion model. Seven clades in the phylogeny had an ancestor with cyanobacteria. Reversal to a green algae photobiont is rare. Several switches were estimated through evolutionary time suggesting that there was some flexibility in these traits along the phylogeny; however, close relatives retained cyanobacteria as main photobiont throughout the cyanolichen’s history. Photobiont switches from green algae to cyanobacteria might enhance ecotypical differentiation. These ecotypes could lead to several speciation events in the new lineage resulting in the phylogenetic signal found in this study. We give insights into the origin of lichen diversity exploring the photobiont switch in a phylogenetic context in Pseudocyphellaria s. l. as a model genus.     

Photobiont selectivity for lichens and evidence for a possible glacial refugium in the Ross Sea Region, Antarctica

Lichens are a symbiosis consisting of heterotrophic, fungal (mycobiont) and photosynthetic algal or cyanobacterial (photobiont) components. We examined photobiont sequences from lichens in the Ross Sea Region of Antarctica using the internal transcribed spacer region of ribosomal DNA and tested the hypothesis that lichens from this extreme environment would demonstrate low selectivity in their choice of photobionts. Sequence data from three targeted lichen species (Buellia frigida, Umbilicaria aprina and Umbilicaria decussata) showed that all three were associated with a common algal haplotype (an unnamed Trebouxia species) which was present in all taxa and at all sites, suggesting lower selectivity. However, there was also association with unique, local photobionts as well as evidence for species-specific selection. For example, the cosmopolitan U. decussata was associated with two photobiont species, Trebouxia jamesii and an unnamed species. The most commonly collected lichen (B. frigida) had its highest photobiont haplotype diversity in the Dry Valley region, which may have served as a refugium during glacial periods. We conclude that even in these extreme environments, photobiont selectivity still has an influence on the successful colonisation of lichens. However, the level of selectivity is variable among species and may be related to the ability of some (e.g. B. frigida) to colonise a wider range of habitats.     

The Melanin Biosynthesis Gene from the CA15-1 Strain of Alfalfa Nodule Bacteria: Molecular Analysis and Phylogeny

Identification and analysis of the melanin biosynthesis gene in the Sinorhizobium meliloti CA15-1 strain were carried out. Tn5 mutants, which lost the ability to synthesize melanin, were obtained. Molecular biological analysis of the mepA gene encoding tyrosinase was performed. In contrast to other members of the Rhizobiacea group, the identical structure of the mepA locus was revealed for all strains of nodule bacteria of the Sinorhizobium genus. Phylogenetic analysis indicated that horizontal transfer of the mepA gene occurred in the course of evolution of bacterial tyrosinases. At the same time, the closely related members of nodule bacteria “acquired” this gene from different sources. Analysis of symbiotic properties of the Mep^– mutants of the CA15-1 strain showed that melanin did not affect the ability to go into an efficient symbiosis with alfalfa. Most probably, it is important only at the stages of adaptation of the free-living cells in the environment.     

Symbiotic performance,nitrogen flux and growth of lima bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L.) varieties inoculated with different indigenous strains of rhizobia

Nitrate uptake and nitrogen inclusion into amino acids were studied in the intact thallus and isolated bionts of the lichen Parmelia sulcata with the aid of mass spectroscopic tracing of heavy isotope ¹⁵N. The isolated photobiont, the green algae Trebouxia sp. did not take up nitrate, whereas the mycobiont and intact thalli were enriched in ¹⁵N when incubated with Na¹⁵NO₃. Pulse feeding experiments with intact thalli followed by separation of photobiont showed that the labelled nitrate was originally assimilated by the mycobiont and only after that was detected in the photobiont. The isolated mycobiont after pulse labeling excreted labeled compounds into the incubation medium. Amino acids were detected in the exudate. The quantities of two amino acids considerably exceeded those of the others. One was identified as alanine, the other could not yet be identified with certainty. Both of these high-quantity compounds were also much more enriched in ¹⁵N than the others. These two compounds are proposed to be the transport forms of nitrogen within the Parmelia sulcata thallus.     

Dehydration-induced changes in spectral reflectance indices and chlorophyll fluorescence of Antarctic lichens with different thallus color,and intrathalline photobiont

In this study, we investigated responses of the Photochemical Reflectance Index (PRI), and Normalized Difference Vegetation Index (NDVI) to gradual dehydration of several Antarctic lichen species (chlorolichens: Xanthoria elegans, Rhizoplaca melanophthalma, Physconia muscigena, cyanolichen: Leptogium puberulum), and a Nostoc commune colony from fully wet to a dry state. The gradual loss of physiological activity during dehydration was evaluated by chlorophyll fluorescence parameters. The experimental lichen species differed in thallus color, and intrathalline photobiont. In the species that did not exhibit color change with desiccation (X. elegans), NDVI and PRI were more or less constant (mean of 0.25, ??0.36, respectively) throughout a wide range of thallus hydration status showing a linear relation to relative water content (RWC). In contrast, the species with apparent species-specific color change during dehydration exhibited a curvilinear relation of NDVI and PRI to RWC. PRI decreased (R. melanophthalma, L. puberulum), increased (N. commune) or showed a polyphasic response (P. muscigena) with desiccation. Except for X. elegans, a curvilinear relation was found between the NDVI response to RWC in all species indicating the potential of combined ground research and remote sensing spectral data analyses in polar regions dominated by lichen flora. The chlorophyll fluorescence data recorded during dehydration (RWC decreased from 100 to 0%) revealed a polyphasic species-specific response of variable fluorescence measured at steady state—F_s, effective quantum yield of photosystem II (Φ_PSII), and non-photochemical quenching (qN). Full hydration caused an inhibition of Φ_PSII in N. commune while other species remained unaffected. The dehydration-dependent fall in Φ_PSII was species-specific, starting at an RWC range of 22–32%. Critical RWC for Φ_PSII was around 5–10%. Desiccation led to a species-specific polyphasic decrease in F_s and an increase in qN indicating the involvement of protective mechanisms in the chloroplastic apparatus of lichen photobionts and N. commune cells. In this study, the spectral reflectance and chlorophyll fluorescence data are discussed in relation to the potential of ecophysiological processes in Antarctic lichens, their resistance to desiccation and survival in Antarctic vegetation oases.     

Diversity and distribution of cultured endolichenic fungi in the Ny-Ålesund Region,Svalbard (High Arctic)

Endolichenic fungi within 17 lichen species in the area near Ny-Ålesund (Svalbard, High Arctic) were studied by a culture-based method. The 247 fungal isolates were obtained from 2712 lichen thallus segments. The colonization rate of endolichenic fungi ranged from 1.6 to 26.5 %, respectively. These isolates were identified to 40 fungal taxa, including 35 Ascomycota (10 orders), 4 Basidiomycota (3 orders), and 1 unidentified fungus. Thelebolales was the most abundant order, while Sordariales were the most diverse order. The common fungal taxa shared by more than 3 lichen species were Thelebolus microsporus (93 isolates), Coniochaeta hoffmannii (7 isolates), Sarocladium kiliense (33 isolates), Coniochaeta sp. 1 (5 isolates), Coniochaeta sp. 4 (28 isolates), and Coniochaeta sp. 2 (5 isolates). Low Sorenson’s similarity coefficients were observed among different lichen species, indicating that host-related factor may shape the endolichenic fungal communities in this region. In addition, no endolichenic fungal taxa were previously found in the Antarctica and Austrian Alps, suggesting endolichenic fungal communities in this region might be also shaped by the Arctic climate. The results demonstrate the existence of specific cultured endolichenic fungal species, which may be suitable objects for further study of their possible functional roles in the lichen thalli.     

Nitrate uptake by isolated bionts of the lichen <Emphasis Type="Italic">Parmelia sulcata</Emphasis>

It was found that bionts isolated from the lichen Parmelia sulcata Taylor had a marked difference with respect to nitrate assimilation. Isolated and purified photobiont, the green alga Trebouxia sp. showed no ability of nitrate absorption. Mycobiont and thallus fragments containing both bionts absorbed nitrate. Illumination had no essential influence on the rate of nitrate uptake. The respiratory inhibitor sodium azide decreased the rate of nitrate uptake by 80–100%, whereas the photosynthetic inhibitor dichlorophenyldimethylurea did not reduce it. Using mass-spectroscopic technique, it was shown that, in the intact thallus, nitrate was first absorbed by the mycobiont, and only later appeared in the photobiont. Probably such nutritional difference between bionts serves as one of the mechanisms by which the host fungi control the associated green algae and support their symbiosis.     

Distributional and ecophysiological study on the Antarctic lichens species pair <Emphasis Type="Italic">Usnea antarctica</Emphasis>/<Emphasis Type="Italic">Usnea aurantiaco</Emphasis>-<Emphasis Type="Italic">atra</Emphasis>

The factors that control lichen distribution in Antarctica are still not well understood, and in this investigation we focused on the distribution, local and continental, and gas exchange of a species pair, closely related lichens with differing reproductive strategies, Usnea aurantiaco-atra (fertile) and Usnea antarctica (sterile, sorediate). The local distributions of these species were recorded along an altitudinal gradient of nearly 300 m at South Bay, Livingston Island, and microclimate was also recorded over 1 year. The photosynthetic responses to temperature, light and thallus water content were determined under controlled conditions in the laboratory. The species were almost identical in their photosynthetic profiles. Locally, on Livingston Island, U. antarctica was confined to low altitude sites which were warmer and drier, whilst U. aurantiaco-atra was present at all altitudes. This contrasts with its distribution across Antarctica where U. antarctica grows 9° latitude further south than U. aurantiaco-atra. Temperature appears not to be the main controller of distribution in these species, but dryness of habitat, which will influence length of activity periods, may be important.     

Asymmetric Co-evolution in the Lichen Symbiosis Caused by a Limited Capacity for Adaptation in the Photobiont

It is proposed that lichen photobionts, compared to mycobionts, have very limited capacity to evolve adaptations to lichenization, so that the symbionts in lichens do not co-evolve. This is because lichens have (a) no sequential selection of photobiont cells from one lichen into another needed for Darwinian natural selection and (b) no photobiont sexual reproduction in the thallus. Molecular studies of lichen photobionts indicate no predictable patterns of photobiont lineages that occur in lichens so supporting this proposal. Any adaptation by photobionts accumulating beneficial mutations for lichenization is probably insignificant compared to the rate of mycobiont adaptation. This proposal poses questions for research relating the photobiont sexual cycle (genetic and cellular), the fate of photobiont lineages after lichenization, whether lineages of photobionts in thalli change with time, thallus formation by from spores as well as carbohydrate movement from photobionts to mycobionts and regulation of co-development of the symbionts in the thallus.     

Photobiont selectivity leads to ecological tolerance and evolutionary divergence in a polymorphic complex of lichenized fungi

Background and Aims

The integrity and evolution of lichen symbioses depend on a fine-tuned combination of algal and fungal genotypes. Geographically widespread species complexes of lichenized fungi can occur in habitats with slightly varying ecological conditions, and it remains unclear how this variation correlates with symbiont selectivity patterns in lichens. In an attempt to address this question, >300 samples were taken of the globally distributed and ecologically variable lichen-forming species complex Tephromela atra, together with closely allied species, in order to study genetic diversity and the selectivity patterns of their photobionts.

Methods

Lichen thalli of T. atra and of closely related species T. grumosa, T. nashii and T. atrocaesia were collected from six continents, across 24 countries and 62 localities representing a wide range of habitats. Analyses of genetic diversity and phylogenetic relationships were carried out both for photobionts amplified directly from the lichen thalli and from those isolated in axenic cultures. Morphological and anatomical traits were studied with light and transmission electron microscopy in the isolated algal strains.

Key Results

Tephromela fungal species were found to associate with 12 lineages of Trebouxia. Five new clades demonstrate the still-unrecognized genetic diversity of lichen algae. Culturable, undescribed lineages were also characterized by phenotypic traits. Strong selectivity of the mycobionts for the photobionts was observed in six monophyletic Tephromela clades. Seven Trebouxia lineages were detected in the poorly resolved lineage T. atra sensu lato, where co-occurrence of multiple photobiont lineages in single thalli was repeatedly observed.

Conclusions

Low selectivity apparently allows widespread lichen-forming fungi to establish successful symbioses with locally adapted photobionts in a broader range of habitats. This flexibility might correlate with both lower phylogenetic resolution and evolutionary divergence in species complexes of crustose lichen-forming fungi.     

Ideal Osmotic Spaces for Chlorobionts or Cyanobionts Are Differentially Realized by Lichenized Fungi

Lichens result from symbioses between a fungus and either a green alga or a cyanobacterium. They are known to exhibit extreme desiccation tolerance. We investigated the mechanism that makes photobionts biologically active under severe desiccation using green algal lichens (chlorolichens), cyanobacterial lichens (cyanolichens), a cephalodia-possessing lichen composed of green algal and cyanobacterial parts within the same thallus, a green algal photobiont, an aerial green alga, and a terrestrial cyanobacterium. The photosynthetic response to dehydration by the cyanolichen was almost the same as that of the terrestrial cyanobacterium but was more sensitive than that of the chlorolichen or the chlorobiont. Different responses to dehydration were closely related to cellular osmolarity; osmolarity was comparable between the cyanolichen and a cyanobacterium as well as between a chlorolichen and a green alga. In the cephalodium-possessing lichen, osmolarity and the effect of dehydration on cephalodia were similar to those exhibited by cyanolichens. The green algal part response was similar to those exhibited by chlorolichens. Through the analysis of cellular osmolarity, it was clearly shown that photobionts retain their original properties as free-living organisms even after lichenization.Lichens are ubiquitously found in all terrestrial environments, including those with extreme climates such as Antarctica and deserts; they are pioneer organisms in primary succession (Longton, 1988; Ahmadjian, 1993). Colonization ability is largely owed to lichens’ extreme tolerance for desiccation (Ahmadjian, 1993). Although lichens harbor photosynthetic green algae or cyanobacteria (blue-green algae) within their thalli, they show metabolic activity even when dried at 20°C and under conditions of 54% relative humidity (Cowan et al., 1979). This desiccation tolerance partially results from drought resistance originally exhibited by the photobiont. It is further strengthened by lichen symbiosis (Kosugi et al., 2009). Cyanolichens (symbiosis between a fungus and a cyanobacterium) are desiccation-tolerant organisms that favor humid and shady environments, whereas chlorolichens (symbiosis between a fungus and a green alga) tolerate dry and high-light environments (James and Henssen, 1976; Lange et al., 1988). Chlorolichens can perform photosynthesis when the surrounding humidity is high, but cyanolichens require some water in a liquid state (Lange et al., 1986, 2001; Nash et al., 1990; Ahmadjian, 1993).Most poikilohydric photosynthetic organisms can tolerate rapid drying. Biological activity during desiccation and recovery following drought are scarcely affected by protein synthesis inhibitors (Proctor and Smirnoff, 2000). Moderate drought tolerance is attained by increasing compatible solutes (amino acids, sugars, and sugar alcohols) as protective agents during drought stress (Mazur, 1968; Parker, 1968; Hoekstra et al., 2001). An increase in compatible solutes prevents water loss or increases water uptake from the air when humidity is high (Lange et al., 1988). It has been observed, however, that the intracellular solute concentration is low (corresponding to a sorbitol concentration of approximately 0.22 m) in the desiccation-tolerant terrestrial cyanobacterium Nostoc commune (Satoh et al., 2002; Hirai et al., 2004). N. commune photosynthetic activity is lost when incubated in low sorbitol concentrations (Hirai et al., 2004), whereas a Trebouxia spp. chlorobiont freshly isolated from the desiccation-tolerant chlorolichen Ramalina yasudae remains active under the same conditions (Kosugi et al., 2009).Different solute concentrations in photobionts may dictate habitat preferences for chlorolichens and cyanolichens (James and Henssen, 1976; Lange et al., 1988). One might expect that the ideal cellular osmotic pressure (or cellular solute concentration) of a lichenized fungus is problematic, as both the fungus and the photobiont are closely associated in the thallus (Kranner et al., 2005). Thus, we may be able to further hypothesize that the solute concentration itself in original photobionts determines the nature of desiccation tolerance in chlorolichens and cyanolichens.To better understand symbiosis in lichens, it is important to examine how the cellular osmotic pressures of both symbionts contribute to lichen photosynthesis. In this study, cellular osmotic pressures of lichens and photobionts were determined by assessing water potential. The cephalodia-possessing lichen Stereocaulon sorediiferum was chosen as a desiccation-tolerant model organism because it separately harbors a green alga and a cyanobacterium in different compartments of the lichen body. The green algal photobiont is contained in the stem- and branch-like structures, whereas the cyanobacterial photobiont (cyanobiont) is contained in the organism’s cephalodia. For comparison, several chlorolichens (R. yasudae, Parmotrema tinctorum, and Graphis spp.), cyanolichens (Collema subflaccidum and Peltigera degenii), green algae (Prasiola crispa, Trebouxia spp., and Trentepohlia aurea), and cyanobacteria (N. commune, Scytonema spp., and Stigonema spp.) were also analyzed (Fig. 1). The cyanobiont of C. subflaccidum is closely related to N. commune (Ahmadjian, 1993), and the cyanobiont of S. sorediiferum belongs to the genus Stigonema (Kurina and Vitousek, 1999). Green algal photobionts of R. yasudae and S. sorediiferum are Trebouxia spp. (Bergman and Huss-Danell, 1983). For the measurements of water potential, we had to use specimens larger than 0.1 g dry weight for one measurement. Furthermore, the specimens should cover approximately 70% of the surface area of a sample cup with 4 cm diameter that was equipped in our dewpoint potentiometer. Considering the statistical analyses, we needed large amounts of lichen and algal samples for the measurement of water potential. To conduct this study, we wanted to use free-living green algae and cyanobacteria, not the photobionts isolated from a lichen body. This is because inconsistent results were reported previously for chlorobionts liberated from lichens (Brock, 1975; Lange et al., 1990). Three major photobionts of lichens, Trebouxia, Trentepohlia, and Nostoc spp., were considered for inclusion. Until now, free-living Trebouxia spp. were not observed convincingly in nature. Therefore, cultivated Trebouxia spp. were used. Other green algae and cyanobacteria were chosen from among free-living species that (1) are closely related to some photobionts, (2) form large communities sufficient to cover the required quantity that will not destroy the local ecosystem by our sampling, (3) are easy to remove from other attached algae/microorganisms, and (4) are tolerant to desiccation. P. crispa forms large communities in nature, and the closely related species Prasiola borealis is known to be a photobiont of Mastodia tessellata. Only two freshwater species of genus Prasiola are found in Japan; P. crispa inhabits a limited area of Hokkaido Island, and Prasiola japonica is a rare species. P. crispa harvested in Antarctica and shown to be desiccation tolerant in our previous work (Kosugi et al., 2010b) was used in this study.Open in a separate windowFigure 1.Lichens analyzed in this study. A, Cyanolichen C. subflaccidum on a rock. B, Wet (left) and dry (right) thalli of cyanolichen Peltigera degenii with green moss. C, Chlorolichen R. yasudae on a rock. D, Chlorolichen Graphis spp. on a Zelkova serrata tree trunk. The grayish basal part of Graphis spp. is the site where the photobiont resides, and the dark-colored streaks are the apothecia. E, Chlorolichen Parmotrema tinctorum on a Z. serrata tree trunk. F, Cephalodia-possessing lichen S. sorediiferum. Some cephalodia are indicated by arrows. The stem- and branch-like structures are the green algae-containing compartments.     

Changes in the lichen biota of the Lions Rump area,King George Island,Antarctica, over the last 20 years

Climate changes observed in recent years in the maritime Antarctic have affected the tundra vegetation, including plant communities in which lichens are a dominant component. The results of comparative studies (1988 and 1990 vs. 2007 and 2008) on the dynamics of the lichen biota within the Antarctic Specially Protected Area No. 151 (King George Island, Antarctica) minimally influenced by human impact, are presented. This long-term experiment is aimed at determining the trends and rate of changes on lichen biota induced by climate warming and rapid deglaciation. The most significant changes affecting the lichen biota have taken place in the forefield of a glacier and on the young moraines where in the second period of studies three species (Polyblastia gothica, Thelenella kerguelena, Thelocarpon cyaneum) were not refound. There was also a reduction in the number of other sites for some species (e.g. Leptogium puberulum, Staurothelle gelida) caused by substrate desiccation. On the other hand, there was an increase in the range of pioneering species (e.g. Bacidia chrysocolla, Caloplaca johnstonii, Candelariella aurella, Lecanora dispersa) on young moraines recently uncovered by the retreating glacier. The smallest changes were observed on the cliff rocks near penguin colonies.     

Aseptic rearing procedure for the stinkbug <Emphasis Type="Italic">Plautia stali</Emphasis> (Hemiptera: Pentatomidae) by sterilizing food-derived bacterial contaminants

The stinkbug Plautia stali Scott is a notorious agricultural pest whose posterior midgut hosts specific bacteria essential for its growth and survival, highlighted as an experimental model for symbiosis studies. Some symbiotic bacteria of P. stali are cultivable, found free-living in and acquired from the environment, and, furthermore, some free-living environmental bacteria are potentially capable of establishing symbiotic association with P. stali. In this context, it is expected that such environmental bacteria may occasionally contaminate and infect the experimental insects maintained in the laboratory, which could potentially affect the functional analyses of the symbiosis. Here we report that such contamination events do occur under a laboratory rearing conditions for P. stali. When symbiont-deprived newborn nymphs from surface-sterilized eggs were reared in sterilized plastic containers with autoclaved water, most of them died as nymphs presumably as a result of aposymbiosis, but only a small fraction could attain adulthood and the adult insects were all infected with γ-proteobacteria allied to Pantoea and Enterobacter. A variety of bacteria, mainly Bacillus and also Pantoea and Enterobacter, were detected from peanuts and soybeans provided as food for P. stali. Autoclaving of peanuts and soybeans eradicated these bacteria but negatively affected the host survival, whereas ethanol sterilization of peanuts and soybeans removed Pantoea and Enterobacter, but not Bacillus, without negative effects on the host survival. On the basis of these results, we established a practical procedure for aseptic rearing of P. stali, which will enable reliable and strict analyses of host–symbiont interactions in the model symbiotic system.     

In situ monitoring of microclimate and metabolic activity in lichens from Antarctic extremes: a comparison between South Shetland Islands and the McMurdo Dry Valleys

Lichens are the dominant organisms in terrestrial Antarctic ecosystems and show a decline in species number, coverage, and growth rate from the maritime Antarctic (62°S) to the McMurdo Dry Valleys (78°S). While Livingston Island (maritime Antarctica) is a hot spot for lichen biodiversity, the McMurdo Dry Valleys (continental Antarctica) are known as one of the most extreme environments for life. Previous studies suggest the biodiversity gradient to be linked to water availability acting through length of active period, but no activity data are available for the Dry Valleys. The work presented here compares metabolic activity of lichens at Livingston Island and the Dry Valleys for 4½ months from continuous monitoring that involves concurrent measurements of chlorophyll fluorescence and microclimate. The latitudinal comparison involves two contrasting habitats for plant physiological activity and microclimate. Two species of the foliose genus Umbilicaria were monitored in both regions plus one sample of the crustose Caloplaca in the Dry Valleys. The results showed a very large difference in the duration of activity over the monitoring period, and this supports the different coverage, species abundance, and growth rates already reported for lichens between both regions. Despite this large difference in activity, and in habitat conditions, analysis of the activity behaviour of the two Umbilicaria species shows interesting common features, while the crustose Caloplaca had additional strategies to improve hydration. This offers one explanation for the abundance of crustose lichens inside the Valleys, indicating better adaptation strategies to a polar desert.     

Phosphate Limitation Alters <Emphasis Type="Italic">Medicago–Sinorhizobium</Emphasis> Signaling: Flavonoid Synthesis and AHL Production

The legume plant Medicago truncatula Gaertn. can establish a symbiotic interaction with Sinorhizobium meliloti. One of the most limiting factors for symbiosis is phosphate (P) deficiency. Therefore, legumes and their symbiotic partners, rhizobia, have developed mechanisms to adapt to P restriction. In the non-symbiotic state, plants would up-regulate flavonoid biosynthesis via increasing the expression of chalcone synthase (chs), catalyzing the first step of flavonoid synthesis. Simultaneously, bacterial quorum sensing (QS) pathway can regulate the expression of certain genes involved in symbiotic functions of bacteria in response to P availability as well as bacterial population. Since both flavonoids and QS signaling molecules (N-acyl homoserine lactones, AHL) play important roles in the rhizobia-legume symbiosis, we evaluated these processes in the symbiotic state under different P concentrations and bacterial populations. In this study, by using real-time PCR and HPLC, we showed the expression of pt1 (phosphate transporter 1) and chs as well as luteolin production increased, in a time dependent manner, in plants following P limitation. Nod gene inducing flavonoids can up-regulate the bacterial QS pathway which results in an increase in AHL production, possibly to enhance symbiotic behaviors of rhizobia. It has been estimated that there is a feedback loop from bacterial AHL to flavonoid production pathway in legume plants.     

Molecular evidence of symbiotic activity between <Emphasis Type="Italic">Symbiodinium</Emphasis> and <Emphasis Type="Italic">Tridacna maxima</Emphasis> larvae

Dinoflagellates in the genus Symbiodinium (zooxanthellae) provide the photosynthesis that sustains the majority of primary production in coral reefs. They occur symbiotically with several phyla, including mollusks such as giant clams (Tridacna spp.). This mutualistic association is obligatory for the giant clams, but the exact point in which this symbiosis is established and the main translocated photosynthate are unknown. In this study, we tracked the expression of specific genes for symbiosis and glycerol synthesis during a time course experiment. Giant clam larvae were raised until 75 h post-fertilization and then infected with cultured isolates of Symbiodinium clade A3. Expression of symbiosis-specific and housekeeping genes was monitored at four time points. The expression of H⁺-ATPase, a symbiosis-specific gene in Symbiodinium, was observed at 24 h after symbiont acquisition by the clam larvae. The expression of an enzyme responsible for glycerol synthesis was also observed. Together, these results show that the symbiotic relationship was already in place 24 h after Symbiodinium acquisition, during veliger larval stage. This is the first report using a molecular symbiosis-specific marker that supports symbiotic activity between Symbiodinium and a metazoan larva of an organism that acquires symbionts horizontally. From the expression of the glycerol-synthesizing gene, it was qualitatively determined that Symbiodinium cells may produce glycerol regardless of whether they are free-living or in symbiosis.     

Arbuscular mycorrhizal symbiosis and methyl jasmonate avoid the inhibition of root hydraulic conductivity caused by drought

Hormonal regulation and symbiotic relationships provide benefits for plants to overcome stress conditions. The aim of this study was to elucidate the effects of exogenous methyl jasmonate (MeJA) application on root hydraulic conductivity (L) of Phaseolus vulgaris plants which established arbuscular mycorrhizal (AM) symbiosis under two water regimes (well-watered and drought conditions). The variation in endogenous contents of several hormones (MeJA, JA, abscisic acid (ABA), indol-3-acetic acid (IAA), salicylic acid (SA)) and the changes in aquaporin gene expression, protein abundance and phosphorylation state were analyzed. AM symbiosis decreased L under well-watered conditions, which was partially reverted by the MeJA treatment, apparently by a drop in root IAA contents. Also, AM symbiosis and MeJA prevented inhibition of L under drought conditions, most probably by a reduction in root SA contents. Additionally, the gene expression of two fungal aquaporins was upregulated under drought conditions, independently of the MeJA treatment. Plant aquaporin gene expression could not explain the behaviour of L. Conversely, evidence was found for the control of L by phosphorylation of aquaporins. Hence, MeJA addition modified the response of L to both AM symbiosis and drought, presumably by regulating the root contents of IAA and SA and the phosphorylation state of aquaporins.     

Taxonomic and functional diversity in <Emphasis Type="Italic">Calogaya</Emphasis> (lichenised Ascomycota) in dry continental Asia

The genus Calogaya (Teloschistaceae, Xanthorioideae) was established to accommodate mainly epilithic lichens with lobate thalli, previously regarded as the “Caloplaca saxicola group.” Data supporting the recognition of this new genus came from European lichens, and although the genus is soundly based, we have found in Asia numerous epiphytic lineages and lineages with reduced, non-lobate thallus in dry continental areas. The taxonomic and functional diversity of Calogaya is distinctly higher in steppe and desert areas of Asia than in the less arid regions of Europe. We sampled 238 specimens, mostly from arid regions of north-western China, Iran, southern Siberia and Turkey. Three nuclear DNA loci were analysed separately and jointly by Bayesian inference, maximum likelihood and *BEAST approaches. Delimitations of 28 putative species were tested by BP&P multispecies coalescent model with joint analysis of species delimitation and species-tree estimation. Finally, we recognised 22 taxonomic units: 16 are at species rank, 3 are treated as subspecies and 3 are complexes, treated here as a single entity, but in reality probably including more than one species. Calogaya altynis, C. biatorina subsp. asiatica, C. decipiens subsp. esorediata, C. haloxylonis, C. orientalis, C. xanthoriella and C. xinjiangis are newly described. Caloplaca zoroasteriorum is combined into Calogaya, and Calogaya persica is reduced to a subspecies. The taxonomic status of Calogaya saxicola is unclear, and the name is employed here “sensu lato” for several non-monophyletic epilithic lineages with short-lobed thalli. Calogaya biatorina and C. ferrugineoides are the two other heterogeneous taxonomic units probably including more species.     

Sensitivity of Photosystem 2 of Antarctic Lichens to High Irradiance Stress: Fluorometric Study of Fruticose (Usnea antarctica) and Foliose (Umbilicaria decussata) Species

Two lichen species collected in maritime Antarctica (King George Island) were exposed under laboratory conditions to excess irradiance to evaluate the response of photosystem 2 (PS2). The response was measured on fully hydrated lichen thalli at 5 °C by means of a modulated fluorometer using chlorophyll (Chl) fluorescence induction curve supplemented with analysis of quenching mechanisms. Chl fluorescence parameters [i.e. ratio of variable to maximum Chl fluorescence (F_V/F_M), quantum yield of PS2 photochemical reactions (₂), quenching coefficients] were evaluated before and several times after exposition to high irradiance in order to characterise the extent of photoinhibition, fast and slow phase of recovery. Strong irradiance (2 000 mol m^–2 s^–1) caused high degree of photoinhibition, particularly higher in fruticose (Usnea antarctica) than in foliose (Umbilicaria decussata) lichen species. Fast phase of recovery from photoinhibition, corresponding to regulatory mechanisms of PS2, was more apparent in U. decussata and ₂ than in U. antarctica and F_V/F_M and ₂ within 40 min after photoinhibitory treatment. It was followed by a slow phase lasting several hours, corresponding to repair and re-synthesis processes. After photoinhibitory treatment, recovery of non-photochemical quenching (NPQ) was faster and more pronounced in U. decussata than in U. antarctica. Significant differences were found between the two species in the rate of recovery in fast-(q_E) and slow-recovering (q_T+I) component of NPQ.     

Breaking the language barrier: experimental evolution of non-native <Emphasis Type="Italic">Vibrio fischeri</Emphasis> in squid tailors luminescence to the host

Although most Vibrio fischeri isolates are capable of symbiosis, the coevolution of certain strains with the Hawaiian bobtail squid, Euprymna scolopes, has led to specific adaptation to this partnership. For instance, strains from different hosts or from a planktonic environment are ineffective squid colonists. Even though bioluminescence is a symbiotic requirement, curiously, symbionts of E. scolopes are dim in culture relative to fish symbionts and free-living isolates. It is unclear whether this dim phenotype is related to the symbiosis or simply coincidental. To further explore the basis of symbiont specificity, we developed an experimental evolution model that utilizes the daily light organ venting behavior of the squid and horizontal acquisition of symbionts for serial passage of cultures. We passaged six populations each derived from the squid-naïve strains of V. fischeri MJ11 (a fish symbiont) and WH1 (a free-living isolate) through a series of juvenile squid light organs. After 15 serially colonized squid for each population, or an estimated 290–360 bacterial generations, we isolated representatives of the light organ populations and characterized their bioluminescence. Multiple evolved lines of both strains produced significantly less bioluminescence both in vitro and in vivo. This reduction in bioluminescence did not correlate with reduced quorum sensing for most isolates tested. The remarkable phenotypic convergence with squid symbionts further emphasizes the importance of bioluminescence in this symbiosis, and suggests that reduced light production is a specific adaptation to the squid.