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.     

Rockin' in rhythm,growth dynamics of the peat moss Sphagnum

The genus Sphagnum is an essential component in the formation and maintenance of high latitude peatlands, bogs and mires. The species grows in dense, extended mats of agglomerated shoots that allow it to retain water necessary for its growth. These mats are partly responsible for maintaining the right conditions for other species in these wetlands to thrive. In this issue of Physiologia Plantarum, Mironov et al. (2020) monitored the growth of Sphagnum riparium for a period of 4 years and revealed three distinct growth rhythms: a seasonal temperature dependent, a circalunar and a third one, synchronized with the circalunar. This synchronised nature of Sphagnum growth could contribute to its position as a key species in the maintenance of peatlands.     

North American origin and recent European establishments of the amphi-Atlantic peat moss Sphagnum angermanicum

Genetic and morphological similarity between populations separated by large distances may be caused by frequent long-distance dispersal or retained ancestral polymorphism. The frequent lack of differentiation between disjunct conspecific moss populations on different continents has traditionally been explained by the latter model, and has been cited as evidence that many or most moss species are extremely ancient and slowly diverging. We have studied intercontinental differentiation in the amphi-Atlantic peat moss Sphagnum angermanicum using 23 microsatellite markers. Two major genetic clusters are found, both of which occur throughout the distributional range. Patterns of genetic structuring and overall migration patterns suggest that the species probably originated in North America, and seems to have been established twice in Northern Europe during the past 40,000 years. We conclude that similarity between S. angermanicum populations on different continents is not the result of ancient vicariance and subsequent stasis. Rather, the observed pattern can be explained by multiple long-distance dispersal over limited evolutionary time. The genetic similarity can also partly be explained by incomplete lineage sorting, but this appears to be caused by the short time since separation. Our study adds to a growing body of evidence suggesting that Sphagnum, constituting a significant part of northern hemisphere biodiversity, may be more evolutionary dynamic than previously assumed.     

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.     

Microclimate and production of peat moss Sphagnum palustre L. in the warm‐temperate zone

Sphagnum palustre L. is one of the few Sphagnum species distributed in the warm‐temperate zone. To elucidate the mechanisms that enable S. palustre to maintain its productivity under warm climatic conditions, we examined the temperature conditions and photosynthetic characteristics of this species in a lowland wetland in western Japan. Moss temperatures during the daytime were much lower than the air temperature, particularly during summer. The optimum temperature for the net photosynthetic rate was approximately 20°C, irrespective of the season, but summer and autumn samples maintained high rates at higher temperatures as well. The net photosynthetic rate at near light saturation was much higher during summer–autumn than during spring–winter. A model estimation in which net production was calculated from the photosynthetic characteristics and microclimatic data showed that both the low temperature of the moss colony and the seasonal shift in photosynthetic characteristics are among the mechanisms that enable this species to maintain its productivity under warm climatic conditions.     

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.     

Hybridization between the peat mosses, Sphagnum capillifolium and S. quinquefarium (Sphagnaceae, Bryophyta) as inferred by morphological characters and isozyme markers

 Isozyme markers and morphological characters were studied in four populations of Sphagnum capillifolium and S. quinquefarium. Recombinant plants were found in three populations, where the two species occur sympatrically. All recombinants possessed different haplotypes and combinations of morphological characters, which show that they are results of independent hybridization events. Strongly male-biased sex ratios were found for Sphagnum capillifolium in all populations where it grew sympatrically with S. quinquefarium. Most of the recombinants were also male fertile. These observations suggest that S. quinquefarium is the female parent in the primary crosses and in subsequent backcrosses. Received September 3, 2001; accepted March 16, 2002 Published online: November 7, 2002 Addresses of the authors: Nils Cronberg (e-mail: Nils.Cronberg@sysbot.lu.se), Department of Systematic Botany, Lund University, S?lvegatan 37, SE-223 62 Lund, Sweden. Rayna Natcheva (e-mail: renimoss@iph.bio.bas.bg), Institute of Botany, Bulgarian Academy of Sciences, 23 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria.     

Freezing and high temperature thresholds of photosystem 2 compared to ice nucleation, frost and heat damage in evergreen subalpine plants

Freezing and high temperature thresholds of photosystem 2 (PS2), ice formation and frost and heat damage were measured in leaves of evergreen subalpine plants under conditions of naturally low (winter) to high (summer) PS2 efficiencies (F_V/F_M). The temperature‐dependent change in basic Chl fluorescence (F₀) (T‐F₀) technique that is usually used to assess the high temperature threshold of PS2 in a new approach was applied to test freezing temperature thresholds of PS2. T‐F₀ curves (+5 °C to ?10 °C at 2 K h^?1) revealed a significant, sudden increase in F₀ on extracellular ice formation (?4.0 or ?5.5 °C). The rise in F₀ was recorded 0.3–0.6 K below ice nucleation (10–20 min later) and was produced by freeze dehydration of cells. The rise in F₀ was not caused by frost damage, as during winter LT₅₀ was lower than ?27 °C and not by formation of ice on the leaf surface. Hence, F₀ measurements during freezing are a useful tool to distinguish between surface ice and extracellular ice inside the leaf tissue which cannot be differentiated by other ice‐detecting methods. PS2 efficiency significantly affected the shape of the high temperature T‐F₀ curves (20–65 °C at 1 K min^?1). Under F_V/F_M >0.6, two F₀ maxima were recorded. The fast rise phase to the first F₀ maximum corresponded with tissue heat damage (LT₅₀: 46.9–54.3 °C). The second F₀ maximum occurred at leaf temperatures between 55 and 60 °C. Under F_V/F_M <0.2 only, the second F₀ maximum was detectable. Lack of awareness of the missing F₀ maximum would lead to an overestimation of the PS2 high temperature threshold by >10 K; hence, under low F_V/F_M, it cannot be determined by the T‐F₀ technique.     

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.     

Climate drivers alter nitrogen availability in surface peat and decouple N2 fixation from CH4 oxidation in the Sphagnum moss microbiome

Peat mosses (Sphagnum spp.) are keystone species in boreal peatlands, where they dominate net primary productivity and facilitate the accumulation of carbon in thick peat deposits. Sphagnum mosses harbor a diverse assemblage of microbial partners, including N₂-fixing (diazotrophic) and CH₄-oxidizing (methanotrophic) taxa that support ecosystem function by regulating transformations of carbon and nitrogen. Here, we investigate the response of the Sphagnum phytobiome (plant + constituent microbiome + environment) to a gradient of experimental warming (+0°C to +9°C) and elevated CO₂ (+500 ppm) in an ombrotrophic peatland in northern Minnesota (USA). By tracking changes in carbon (CH₄, CO₂) and nitrogen (NH₄-N) cycling from the belowground environment up to Sphagnum and its associated microbiome, we identified a series of cascading impacts to the Sphagnum phytobiome triggered by warming and elevated CO₂. Under ambient CO₂, warming increased plant-available NH₄-N in surface peat, excess N accumulated in Sphagnum tissue, and N₂ fixation activity decreased. Elevated CO₂ offset the effects of warming, disrupting the accumulation of N in peat and Sphagnum tissue. Methane concentrations in porewater increased with warming irrespective of CO₂ treatment, resulting in a ~10× rise in methanotrophic activity within Sphagnum from the +9°C enclosures. Warming's divergent impacts on diazotrophy and methanotrophy caused these processes to become decoupled at warmer temperatures, as evidenced by declining rates of methane-induced N₂ fixation and significant losses of keystone microbial taxa. In addition to changes in the Sphagnum microbiome, we observed ~94% mortality of Sphagnum between the +0°C and +9°C treatments, possibly due to the interactive effects of warming on N-availability and competition from vascular plant species. Collectively, these results highlight the vulnerability of the Sphagnum phytobiome to rising temperatures and atmospheric CO₂ concentrations, with significant implications for carbon and nitrogen cycling in boreal peatlands.     

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.     

Experimental warming alters the community composition,diversity, and N2 fixation activity of peat moss (Sphagnum fallax) microbiomes

Sphagnum‐dominated peatlands comprise a globally important pool of soil carbon (C) and are vulnerable to climate change. While peat mosses of the genus Sphagnum are known to harbor diverse microbial communities that mediate C and nitrogen (N) cycling in peatlands, the effects of climate change on Sphagnum microbiome composition and functioning are largely unknown. We investigated the impacts of experimental whole‐ecosystem warming on the Sphagnum moss microbiome, focusing on N₂ fixing microorganisms (diazotrophs). To characterize the microbiome response to warming, we performed next‐generation sequencing of small subunit (SSU) rRNA and nitrogenase (nifH) gene amplicons and quantified rates of N₂ fixation activity in Sphagnum fallax individuals sampled from experimental enclosures over 2 years in a northern Minnesota, USA bog. The taxonomic diversity of overall microbial communities and diazotroph communities, as well as N₂ fixation rates, decreased with warming (p < 0.05). Following warming, diazotrophs shifted from a mixed community of Nostocales (Cyanobacteria) and Rhizobiales (Alphaproteobacteria) to predominance of Nostocales. Microbiome community composition differed between years, with some diazotroph populations persisting while others declined in relative abundance in warmed plots in the second year. Our results demonstrate that warming substantially alters the community composition, diversity, and N₂ fixation activity of peat moss microbiomes, which may ultimately impact host fitness, ecosystem productivity, and C storage potential in peatlands.     

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.     

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.