Development of microsatellites for the peat moss Sphagnum capillifolium using ISSR cloning

Sphagnum mosses are major components of peat bogs but populations of many species are under threat due to habitat fragmentation resulting from the cutting of peat for fuel. We have used an intersimple sequence repeat (ISSR)‐based cloning method to develop nine polymorphic nuclear microsatellites for the peat moss species Sphagnum capillifolium. Between three and seven alleles per locus were detected in a sample of 48 haploid gametophytes and levels of gene diversity ranged from 0.5391 to 0.7960. These represent the first microsatellite markers developed for this important genus and most also exhibited cross‐species amplification across a range of common Sphagnum species.     

Recombination and introgression of nuclear and chloroplast genomes between the peat mosses, Sphagnum capillifolium and Sphagnum quinquefarium

Haploid hybrid gametophytes are often present at low frequencies in sympatric populations of Sphagnum capillifolium and Sphagnum quinquefarium. We used intersimple sequence repeat (ISSR) markers and polymerase chain reaction-restriction fragment length polymorphism of the trnL(UAA) intron of the chloroplast genome to reveal the nuclear and chloroplast composition of mature hybrid gametophytes from natural populations and of gametophytes derived from spores of hybrid sporophytes collected in nature. Asymmetrical nuclear inheritance was found in the progeny of the hybrid sporophytes, indicating that only spores with a low level of recombination of parental genomes were viable. A similarly skewed nuclear composition was found among the naturally occurring hybrid gametophytes. All hybrid genomes contained a larger proportion of S. capillifolium ISSR markers, combined with only two to five S. quinquefarium markers together with a chloroplast haplotype derived from S. quinquefarium. In this way, a pattern resembling introgression is created within a single generation. Some individuals possessed nuclear genomes typical for S. capillifolium in combination with the chloroplast haplotype of S. quinquefarium, possibly indicating backcrossing. Our results indicate that hybridization between S. capillifolium and S. quinquefarium is relatively common, but the resistance of large parts of the genome against heterospecific genes maintains the genetic distinctness of the species. Further evolutionary and phylogenetic consequences of restricted interspecific gene exchange are discussed.     

The narrow endemic Norwegian peat moss Sphagnum troendelagicum originated before the last glacial maximum

It is commonly found that individual hybrid, polyploid species originate recurrently and that many polyploid species originated relatively recently. It has been previously hypothesized that the extremely rare allopolyploid peat moss Sphagnum troendelagicum has originated multiple times, possibly after the last glacial maximum in Scandinavia. This conclusion was based on low linkage disequilibrium in anonymous genetic markers within natural populations, in which sexual reproduction has never been observed. Here we employ microsatellite markers and chloroplast DNA (cpDNA)-encoded trnG sequence data to test hypotheses concerning the origin and evolution of this species. We find that S. tenellum is the maternal progenitor and S. balticum is the paternal progenitor of S. troendelagicum. Using various Bayesian approaches, we estimate that S. troendelagicum originated before the Holocene but not before c. 80 000 years ago (median expected time since speciation 40 000 years before present). The observed lack of complete linkage disequilibrium in the genome of this species suggests cryptic sexual reproduction and recombination. Several lines of evidence suggest multiple origins for S. troendelagicum, but a single origin is supported by approximate Bayesian computation analyses. We hypothesize that S. troendelagicum originated in a peat-dominated refugium before last glacial maximum, and subsequently immigrated to central Norway by means of spore flow during the last thousands of years.     

Population structure and interspecific differentiation of the peat moss sister speciesSphagnum rubellum andS. capillifolium (Sphagnaceae) in northern Europe

Isozyme electrophoresis was used to study the morphologically similar sister speciesSphagnum rubellum andS. capillifolium from a sample of 1313 plants representing 37 populations from Scandinavia, Great Britain and S Germany. The mean pairwise genetic identities (I) among conspecific populations were 0.976 forS. rubellum and 0.969 forS. capillifolium, versus 0.627 between populations of the two species. Interspecific gene flow was indicated by the observation of occasional plants in sympatric populations with alleles otherwise unique to the other species. Populations of bisexualS. capillifolium were significantly more variable than populations of unisexualS. rubellum. Alpine populations ofS. rubellum andS. capillifolium were dominated by few genotypes, and differentiation among populations was pronounced, indicating a low level of sexual recombination. InS. rubellum, maximum variability was found in western areas with high annual precipitation. Distribution of alleles inS. rubellum indicated restricted gene flow between Great Britain and Scandinavia. Postglacial migration from separate refugia may explain large-scale variation inS. rubellum.     

Seasonal dynamics of N in two Sphagnum moss species and the underlying peat treated with 15NH415NO3

Uptake of 15N labelled NH4NO3 by two Sphagnum mosses on a raised bog in north east Scotland was measured at different times of the year. In a field experiment, fortnightly additions of NH4NO3 at natural abundance, equivalent to 3 g N m-2 yr-1, were made over 14 months to cores of Sphagnum capillifolium occupying hummocks and S. recurvum colonizing hollows. Pre-harvested cores were treated with 15NH415NO3 two weeks before harvesting and 15N abundance determined for the total N in the moss, inorganic and dissolved organic N (DON) in the moss water and extractable inorganic, organic and microbial N in the underlying peat. The proportion of added 15N taken up by the mosses two weeks after each addition averaged 72% and ranged between 11 and 100%, tending to be least during October when the rising water table reached the surface, particularly for S. recurvum. A small proportion of the 15N was detected in the moss water as NH4+ (0.01%) and as DON (0.03%) and on occasions a large proportion remained unaccounted for. In waters from S. capillifolium, DON was proportional to the amount of inorganic N added, but this was not the case for S. recurvum. Little or no 15N was detected in the underlying peat partly because of the large size and variability of the NH4+, DON and microbial N pools.     

The rare peat moss Sphagnum wulfianum (Sphagnaceae) did not survive the last glacial period in northern European refugia

? Premise of the study: Organisms may survive unfavorable conditions either by moving to more favorable areas by means of dispersal or by adapting to stressful environments. Pleistocene glacial periods represent extremely unfavorable conditions for the majority of life forms, especially sessile organisms. Many studies have revealed placements of refugial areas and postglacial colonization patterns of seed plants, but little is still known about areas of long-term survival and historical migration routes of bryophytes. Given overall differences in stress tolerance between seed plants and bryophytes, it is of interest to know whether bryophytes have survived periods of extreme climatic conditions better then seed plants in northern areas. ? Methods: The haploid and rarely spore-producing peat moss Sphagnum wulfianum is mostly found in areas that were covered by ice during the last glacial maximum. Twelve microsatellite markers were amplified from 43 populations (367 shoots) of this species, and data were analyzed using population genetic diversity statistics, Bayesian clustering methods, and coalescence-based inference tools to estimate historical and demographic parameters. ? Key results: Genetic diversity within populations was low, but populations were highly differentiated, with two main genetic clusters being recognized. ? Conclusion: The two main genetic groups have diverged quite recently in the Holocene, and the pattern of genetic variability and structuring gives no support for survival in Scandinavian refugia during the last glacial period in this species. The dispersal ability of this plant thus seems surprisingly high despite its infrequent spore production.     

Impact of NH4NO3 on microbial biomass C and N and extractable DOM in raised bog peat beneath Sphagnum capillifolium and S. recurvum

Regular bi-weekly additions of NH₄NO₃, equivalent to a rate of 3 g N m^–2 yr^–1, were applied to cores of Sphagnum capillifolium, inhabiting hummocks and S. recurvum a pool and hollow colonizer, in a raisedbog in north east Scotland. Microbial biomass C and N,both measured by chloroform extraction, showed similarseasonal patterns and, for most depths, the effects ofadded N on microbial biomass C and N changed withtime. The addition of inorganic N had greatest effectduring October when the water table had risen to thesurface and microbial C and N in the untreated coreshad decreased. Microbial C and N were maintained at75 g C m^–2 and 8.3 g N m^–2 above the values in the untreated cores and far exceeded the amounts of N that had been added up to that date (1 g N m^–2) as NH₄NO₃. This increased microbial biomass was interpreted as leaching of carbonaceous material from the NH₄NO₃ treated moss resulting in greater resistance of the microbialbiomass to changes induced by the rising water table.Treatment with N also caused significant reductions inextractable dissolved organic N (DON) at 10–15 cmdepth, beneath the surface of the moss, but at lowerdepths to 25 cm no changes were observed. Extracteddissolved organic carbon (DOC) was not affected by Ntreatment and showed less seasonal variation than DON,such that the C:N ratio of dissolved organic matter(DOM) in all depths increased from approximately 4 inJuly to around 30 in December.     

Genetic diversity in populations of Sphagnum capillifolium from the mountains of Bulgaria,and their possible refugial role

Abstract

Genetic diversity in eight populations of Sphagnum capillifolium from different Bulgarian mountains was investigated by means of isozyme electrophoresis. High levels of allelic diversity were found (H_S = 0.119), comparable to earlier estimates for northern European populations (H_S = 0.116). Strong differentiation among populations and a low number of widespread genotypes suggest a high degree of isolation and restricted gene flow between populations, which is consistent with generally small and scattered populations. The large proportion of distinguishable genotypes (mean 0.498) suggests high levels of out-crossing either currently or in the past. Introgression between S. capillifolium and S. rubellum, a species not found in Bulgaria, was suggested by the occurrence of rubellum-alleles in five populations from different mountains. This could be explained by an ancient hybridization event in a sympatric population. Based on (1) the high genetic diversity, (2) the fairly wide distribution of alien alleles, and (3) the isolated distribution of populations even within one mountain, a possible survival of S. capillifolium in the Balkan area during the Quaternary ice periods is hypothesized.     

Structural colour in the moss Sphagnum subnitens Russ. & Warnst

Abstract

The blue-grey sheen of dried specimens of Sphagnum subnitens is attributed to thin film interference in the walls of the hyaline cells, this being produced by reflection from upper and lower film surfaces. The reflectance is large in air and small in water, so that the colour is visible only in dried specimens. S. rubellurn lacks the blue-grey sheen because only a small part of the total area of the hyaline cells is thin enough to generate interference colour.     

Tyrosine D oxidation at cryogenic temperature in photosystem II

Photosystem II (PSII) contains two redox-active tyrosines (TyrZ and TyrD) possessing very different properties. TyrD is stable in its oxidized form, while TyrZ is kinetically competent in the water oxidizing reaction. The temperature dependence of the formation of light-induced tyrosyl radical was studied as a function of pH in Mn-depleted PS II from cyanobacteria and plants. Tyrosyl radical formation was observed in the majority of the centers at pH 8.5 and 15 K. The use of two Synechocystis mutants where either TyrZ or TyrD was replaced by a phenylalanine residue allowed a clear demonstration that only TyrD and not TyrZ was oxidized under these conditions. By lowering the pH, the fraction of centers undergoing TyrD oxidation greatly diminished. This pH dependence could be fitted by assuming a single protonable group with a pK(a) of approximately 7.6. This pH dependence correlates with the unexpectedly rapid oxidation of TyrD at room temperature [Faller, P., Debus, R. J., Brettel, K., Sugiura, M., Rutherford, A. W., and Boussac, A. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 14368]. The equally unexpectedly ability to undergo oxidation at cryogenic temperatures reported here, puts limitations on the nature of the protonation reactions associated with electron transfer in this system, raising the possibility of proton tunneling along the hydrogen bond or alternatively the presence of deprotonated TyrD (tyrosinate) prior to oxidation.     

Analysis of the bacterial community developing in the course of Sphagnum moss decomposition

Slow degradation of organic matter in acidic Sphagnum peat bogs suggests a limited activity of organotrophic microorganisms. Monitoring of the Sphagnum debris decomposition in a laboratory simulation experiment showed that this process was accompanied by a shift in the water color to brownish due to accumulation of humic substances and by the development of a specific bacterial community with a density of 2.4 x 10(7) cells ml(-1). About half of these organisms are metabolically active and detectable with rRNA-specific oligonucleotide probes. Molecular identification of the components of this microbial community showed the numerical dominance of bacteria affiliated with the phyla Alphaproteobacteria, Actinobacteria, and Phanctomycetes. The population sizes of Firmicutes and Bacteroidetes, which are believed to be the main agents of bacterially-mediated decomposition in eutrophic wetlands, were low. The numbers of planctomycetes increased at the final stage of Sphagnum decomposition. The representative isolates of Alphaproteobacteria were able to utilize galacturonic acid, the only low-molecular-weight organic compound detected in the water samples; the representatives of Planctomycetes were able to decompose some heteropolysaccharides, which points to the possible functional role of these groups of microorganisms in the community under study. Thus, the composition of the bacterial community responsible for Sphagnum decomposition in acidic and low-mineral oligotrophic conditions seems to be fundamentally different from that of the bacterial community which decomposes plant debris in eutrophic ecosystems at neutral pH.     

Major and trace elements in Sphagnum moss from four southern German bogs,and comparison with available moss monitoring data

In this paper, we present concentrations of an array of major and trace elements (Ag, Al, As, Ba, Bi, Cd, Co, Cr, Cu, Fe, Mn, Mo, Rb, Sb, Sc, Sr, Th, Tl, U, V, Zn) in living Sphagnum mosses from four southern German bogs and compare them with moss monitoring data of the respective regions. To do this, Sphagnum mosses were collected in Upper Bavaria (Oberbayern, OB) and the Northern Black Forest (Nordschwarzwald, NBF). Surfaces of Sphagnum carpets were marked with plastic mesh and, one year later, the annual moss production was harvested. Up to 12 samples (40 cm × 40 cm) were collected per site, and 6–10 sites investigated per bog. The concentrations of these elements were then determined in acid digests using sector field ICP-MS. Variations within a given sampling site were in the range of 2 to 3-fold for all major and trace element concentrations except for Mn (12-fold) and Tl (38-fold). For most of the elements, concentrations between bogs of a given region were significantly different and atmospheric deposition of particles seems to be considerably affected by local circumstances such as tree canopy interception and microtopography. Comparing trace element concentrations measured in Sphagnum mosses for 2007 with published moss monitoring data for 2005 resulted in a very good agreement for most elements. Clearly, Sphagnum mosses from bogs are useful biomonitors for estimating atmospheric contamination by metals. This supports the use of Sphagnum in atmospheric deposition monitoring especially in cases where Sphagnum is abundant (e.g., boreal forests). In regions with neither bogs nor forests, living Sphagnum moss bags could be used to the same effect.     

A few molecules of zeaxanthin per reaction centre of photosystem II permit effective thermal dissipation of light energy in photosystem II of a poikilohydric moss

The relationship between thermal dissipation of light energy (as indicated by the quenching of chlorophyll fluorescence), zeaxanthin availability and protonation reactions was investigated in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. In the absence of zeaxanthin and actinic illumination, acidification by 20% CO₂ in air was incapable of quenching basal, so-called F ₀ fluorescence either in the moss or in spinach (Spinacia oleracea L.) leaves. However, 1-s light pulses given either every 40, 60 or 200 s increased thermal dissipation as indicated by F ₀ and F _m quenching in the presence of 20% CO₂ in air in the moss, but not in spinach while reaction centres of photosystem II (PSII) were photochemically open. In the moss, a few short light pulses, which were separated by prolonged dark times, were sufficient to raise zeaxanthin levels in the presence of 20% CO₂ in air. Simultaneously, quantum efficiency of charge separation in PSII was decreased. Increasing the CO₂ concentration beyond 20% further decreased quantum efficiency even in the absence of short light pulses. Under conditions optimal for fluorescence quenching, one molecule of zeaxanthin per reaction centre of PSII was sufficient to decrease quantum efficiency of charge separation in PSII by 50%. Thus, in combination with a protonation reaction, one molecule of zeaxanthin was as efficient at capturing excitation energy as a photochemically open reaction centre. The data are discussed in relation to the interaction between zeaxanthin and thylakoid protonation, which enables effective thermal dissipation of light energy in the antennae of PSII in the moss but not in higher plants when actinic illumination is absent. Received: 8 April 2000 / Accepted: 31 August 2000     

Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation approaches

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4–2.0 × 10⁷ cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.     

Bacterial populations and environmental factors controlling cellulose degradation in an acidic Sphagnum peat

Northern peatlands represent a major global carbon store harbouring approximately one-third of the global reserves of soil organic carbon. A large proportion of these peatlands consists of acidic Sphagnum-dominated ombrotrophic bogs, which are characterized by extremely low rates of plant debris decomposition. The degradation of cellulose, the major component of Sphagnum-derived litter, was monitored in long-term incubation experiments with acidic (pH 4.0) peat extracts. This process was almost undetectable at 10°C and occurred at low rates at 20°C, while it was significantly accelerated at both temperature regimes by the addition of available nitrogen. Cellulose breakdown was only partially inhibited in the presence of cycloheximide, suggesting that bacteria participated in this process. We aimed to identify these bacteria by a combination of molecular and cultivation approaches and to determine the factors that limit their activity in situ. The indigenous bacterial community in peat was dominated by Alphaproteobacteria and Acidobacteria. The addition of cellulose induced a clear shift in the community structure towards an increase in the relative abundance of the Bacteroidetes. Increasing temperature and nitrogen availability resulted in a selective development of bacteria phylogenetically related to Cytophaga hutchinsonii (94-95% 16S rRNA gene sequence similarity), which densely colonized microfibrils of cellulose. Among isolates obtained from this community only some subdivision 1 Acidobacteria were capable of degrading cellulose, albeit at a very slow rate. These Acidobacteria represent indigenous cellulolytic members of the microbial community in acidic peat and are easily out-competed by Cytophaga-like bacteria under conditions of increased nitrogen availability. Members of the phylum Firmicutes, known to be key players in cellulose degradation in neutral habitats, were not detected in the cellulolytic community enriched at low pH.     

Transfer of fixed-N from N2-fixing cyanobacteria associated with the moss Sphagnum riparium results in enhanced growth of the moss

Background

Despite the general assumption that nitrogen fixed by associated cyanobacteria will be readily utilised for growth by the Sphagnum, no empirical evidence is available in the literature. Therefore the effects of nitrogen transfer from cyanobacteria associated with S. riparium were investigated.

Methods

Cultivation of S. riparium with and without cyanobacteria was performed under laboratory conditions for 57 days.

Results

We show that nitrogen fixation by cyanobacteria associated with Sphagnum mosses, influences moss growth by transfer of fixed nitrogen to the moss. More than 35 % of the nitrogen fixed by cyanobacteria was transferred to the newly formed moss biomass and resulted in an increase in the growth of Sphagnum biomass compared to the controls. The variation in the increase of nitrogen content explained 76 % of the biomass increment.

Conclusion

Hence, nitrogen fixation will have immediate effect on the carbon fixation by Sphagnum. This shows that factors regulating nitrogen fixation will have a direct effect on the role of Sphagnum dominated ecosystems with respect to carbon cycling.     

Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation methods

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4-2.0 x 10(7) cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.