Soil carbon stocks in forest-tundra ecotones along a 500 km latitudinal gradient in northern Norway.

As shrubs and trees are advancing into tundra ecosystems due to climate warming, litter input and microclimatic conditions affecting litter decomposition are likely to change. To assess how the upward shift of high-latitude treeline ecotones might affect soil organic carbon stocks (SOC), we sampled SOC stocks in the surface layers of 14 mountain birch forest-tundra ecotones along a 500 km latitudinal transect in northern Norway. Our objectives were to examine: (1) how SOC stocks differ between forest and tundra soils, and (2) the relative role of topography, vegetation and climate in explaining variability in SOC stock sizes. Overall, forest soils had higher SOC stocks (median: 2.01 kg m-2) than tundra soils (median: 1.33 kg m-2). However, SOC storage varied greatly within and between study sites. Two study sites had higher SOC stocks in the tundra than in the nearby forest, five sites had higher SOC stocks in the forest, and seven sites did not show differences in SOC stocks between forest and tundra soils. Thus, our results suggest that an upwards forest expansion does not necessarily lead to a change in SOC storage at all sites. Further, a partial least-squares regression (PLSR) model indicated that elevation, temperature, and slope may be promising indicators for SOC stock size at high-latitude treelines. Precipitation and vegetation were in comparison only of minor importance.

Phenotypic responses to light, water, and nutrient conditions in the allopolyploid Arabidopsis suecica and its parent species A. thaliana and A. arenosa: Does the allopolyploid outrange its parents?

Polyploid species possess more than two sets of chromosomes and may show high gene redundancy, hybrid vigor, and masking of deleterious alleles compared to their parent species. Following this, it is hypothesized that this makes them better at adapting to novel environments than their parent species, possibly due to phenotypic plasticity. The allopolyploid Arabidopsis suecica and its parent species A. arenosa and A. thaliana were chosen as a model system to investigate relationships between phenotypic plasticity, fitness, and genetic variation. Particularly, we test if A. suecica is more plastic, show higher genetic diversity, and/or have higher fitness than its parent species. Wild Norwegian populations of each species were analyzed for phenotypic responses to differences in availability of nutrient, water, and light, while genetic diversity was assessed through analysis of AFLP markers. Arabidopsis arenosa showed a higher level of phenotypic plasticity and higher levels of genetic diversity than the two other species, probably related to its outbreeding reproduction strategy. Furthermore, a general positive relationship between genetic diversity and phenotypic plasticity was found. Low genetic diversity was found in the inbreeding A. thaliana. Geographic spacing of populations might explain the clear genetic structure in A. arenosa, while the lack of structure in A. suecica could be due to coherent populations. Fitness measured as allocation of resources to reproduction, pointed toward A. arenosa having lower fitness under poor environmental conditions. Arabidopsis suecica, on the other hand, showed tendencies toward keeping up fitness under different environmental conditions.

Discrimination of grass pollen of different species by FTIR spectroscopy of individual pollen grains.

Fourier-transform infrared (FTIR) spectroscopy enables the chemical characterization and identification of pollen samples, leading to a wide range of applications, such as paleoecology and allergology. This is of particular interest in the identification of grass (Poaceae) species since they have pollen grains of very similar morphology. Unfortunately, the correct identification of FTIR microspectroscopy spectra of single pollen grains is hindered by strong spectral contributions from Mie scattering. Embedding of pollen samples in paraffin helps to retrieve infrared spectra without scattering artifacts. In this study, pollen samples from 10 different populations of five grass species (Anthoxanthum odoratum, Bromus inermis, Hordeum bulbosum, Lolium perenne, and Poa alpina) were embedded in paraffin, and their single grain spectra were obtained by FTIR microspectroscopy. Spectra were subjected to different preprocessing in order to suppress paraffin influence on spectral classification. It is shown that decomposition by non-negative matrix factorization (NMF) and extended multiplicative signal correction (EMSC) that utilizes a paraffin constituent spectrum, respectively, leads to good success rates for the classification of spectra with respect to species by a partial least square discriminant analysis (PLS-DA) model in full cross-validation for several species. PLS-DA, artificial neural network, and random forest classifiers were applied on the EMSC-corrected spectra using an independent validation to assign spectra from unknown populations to the species. Variation within and between species, together with the differences in classification results, is in agreement with the systematics within the Poaceae family. The results illustrate the great potential of FTIR microspectroscopy for automated classification and identification of grass pollen, possibly together with other, complementary methods for single pollen chemical characterization.

Combining Chemical Information From Grass Pollen in Multimodal Characterization.

The analysis of pollen chemical composition is important to many fields, including agriculture, plant physiology, ecology, allergology, and climate studies. Here, the potential of a combination of different spectroscopic and spectrometric methods regarding the characterization of small biochemical differences between pollen samples was evaluated using multivariate statistical approaches. Pollen samples, collected from three populations of the grass Poa alpina, were analyzed using Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, surface enhanced Raman scattering (SERS), and matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). The variation in the sample set can be described in a hierarchical framework comprising three populations of the same grass species and four different growth conditions of the parent plants for each of the populations. Therefore, the data set can work here as a model system to evaluate the classification and characterization ability of the different spectroscopic and spectrometric methods. ANOVA Simultaneous Component Analysis (ASCA) was applied to achieve a separation of different sources of variance in the complex sample set. Since the chosen methods and sample preparations probe different parts and/or molecular constituents of the pollen grains, complementary information about the chemical composition of the pollen can be obtained. By using consensus principal component analysis (CPCA), data from the different methods are linked together. This enables an investigation of the underlying global information, since complementary chemical data are combined. The molecular information from four spectroscopies was combined with phenotypical information gathered from the parent plants, thereby helping to potentially link pollen chemistry to other biotic and abiotic parameters.

Spruce and beech as local determinants of forest fungal community structure in litter, humus and mineral soil.

Beech forests reaches its native distribution limit in SE Norway, but is expected to expand substantially northwards due to climate warming. This may potentially result in a fundamental transformation of contemporary Northern European forests, with tentative effects on the associated belowground fungi. Fungal communities mediate vital ecosystem processes such as ecosystem productivity and carbon sequestration in boreal forests. To investigate how soil fungi is affected by the vegetation transition from spruce to beech forest, we sampled litter, humus and mineral soil in a forest landscape dominated by beech, spruce or a mixture of these. The fungal communities in the soil samples were analyzed by DNA metabarcoding of the rDNA ITS2 region. Although soil layers were the most important structuring gradient, we found clear differences in fungal species composition between spruce and beech plots. The differences in fungal community composition were most evident in the litter and least in the mineral soil. Decomposers, most notably Mycena, dominated the litter layer while various mycorrhizal fungi dominated the humus and mineral layers. Some ectomycorrhizal taxa, such as Cenoccocum and Russula, were more abundant in spruce forests. Differences in fungal community composition between forest types can potentially have large impacts on carbon sequestration rates.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) shows adaptation of grass pollen composition.

MALDI time-of-flight mass spectrometry (MALDI-TOF MS) has become a widely used tool for the classification of biological samples. The complex chemical composition of pollen grains leads to highly specific, fingerprint-like mass spectra, with respect to the pollen species. Beyond the species-specific composition, the variances in pollen chemistry can be hierarchically structured, including the level of different populations, of environmental conditions or different genotypes. We demonstrate here the sensitivity of MALDI-TOF MS regarding the adaption of the chemical composition of three Poaceae (grass) pollen for different populations of parent plants by analyzing the mass spectra with partial least squares discriminant analysis (PLS-DA) and principal component analysis (PCA). Thereby, variances in species, population and specific growth conditions of the plants were observed simultaneously. In particular, the chemical pattern revealed by the MALDI spectra enabled discrimination of the different populations of one species. Specifically, the role of environmental changes and their effect on the pollen chemistry of three different grass species is discussed. Analysis of the group formation within the respective populations showed a varying influence of plant genotype on the classification, depending on the species, and permits conclusions regarding the respective rigidity or plasticity towards environmental changes.

Fertilization Changes Chemical Defense in Needles of Mature Norway Spruce (Picea abies).

Nitrogen availability limits growth in most boreal forests. However, parts of the boreal zone receive significant levels of nitrogen deposition. At the same time, forests are fertilized to increase volume growth and carbon sequestration. No matter the source, increasing nitrogen in the boreal forest ecosystem will influence the resource situation for its primary producers, the plants, with possible implications for their defensive chemistry. In general, fertilization reduces phenolic compound concentrations in trees, but existing evidence mainly comes from studies on young plants. Given the role of the phenolic compounds in protection against herbivores and other forest pests, it is important to know if phenolics are reduced with fertilization also in mature trees. The evergreen Norway spruce is long-lived, and it is reasonable that defensive strategies could change from the juvenile to the reproductive and mature phases. In addition, as the needles are kept for several years, defense could also change with needle age. We sampled current and previous year needles from an N fertilization experiment in a Norway spruce forest landscape in south-central Norway to which N had been added annually for 13 years. We analyzed total nitrogen (N) and carbon (C), as well as low-molecular phenolics and condensed tannins. Needles from fertilized trees had higher N than those from controls plots, and fertilization decreased concentrations of many flavonoids, as well as condensed tannins in current year needles. In previous year needles, some stilbenes and condensed tannins were higher in fertilized trees. In control trees, the total phenolic concentration was almost five times as high in previous year needles compared with those from the current year, and there were great compositional differences. Previous year needles contained highest concentrations of acetophenone and stilbenes, while in the current year needles the flavonoids, and especially coumaroyl-astragalins dominated. Condensed tannins did not differ between current and previous year needles from control trees. In conclusion, the phenolic defense of current year needles of mature P.abies trees was strongly changed upon fertilization. This may imply that nitrogen deposition and forest fertilization leave forests less robust in a time when pests may take advantages of a changing climate.

Effect of environmental exposure on charcoal density and porosity in a boreal forest.

Although charcoal's density and porosity shape its environmental roles (e.g. controlling its landscape movement and determining the internal pore space available as microbial habitat), the rate of change of these properties in the environment remains largely unknown. With time, charcoal pores may fill or charcoal particles may shatter, altering the ecosystem services delivered. In this study we examined the effects of environmental exposure on the density and porosity of charcoals pyrolyzed at two different temperatures (350 and 520°C). Fresh charcoal made from the dominant local tree (Pinus sylvestris) was returned in litter incubations to Norwegian boreal forest soils for 20months in three different placements: (i) aboveground, (ii) in the humus layer, and (iii) under the humus layer in contact with the mineral subsoil. By varying soil horizon placement we were able to separate the effects of infill from the effects of environmental disturbance on charcoal density and porosity. Environmental exposure changed charcoal density and porosity, and the response varied with environmental placement. Charcoal placed in soil layers increased in porosity by ~1-2% after 20months' incubation. This is likely because open indentations on the charcoal surface became partially occluded, creating more detectable pore space. In contrast, the porosity of charcoal incubated aboveground decreased slightly (~1-2% over 20months). Because there were no minerals or humic substances to infill the aboveground charcoal samples, this porosity reduction was likely caused by breakage of particles induced by weathering. When charcoal particles cleave through pores, internal pore space is destroyed. The small changes observed here indicate that environmental exposure did not trigger rapid shifts in charcoal density and porosity. In addition, these physical properties appear not to have reached equilibrium after 20months incubation, suggesting that the effect of environmental exposure on charcoal's physical properties occurs on the timescale of years to decades.

A high-throughput FTIR spectroscopy approach to assess adaptive variation in the chemical composition of pollen.

The two factors defining male reproductive success in plants are pollen quantity and quality, but our knowledge about the importance of pollen quality is limited due to methodological constraints. Pollen quality in terms of chemical composition may be either genetically fixed for high performance independent of environmental conditions, or it may be plastic to maximize reproductive output under different environmental conditions. In this study, we validated a new approach for studying the role of chemical composition of pollen in adaptation to local climate. The approach is based on high-throughput Fourier infrared (FTIR) characterization and biochemical interpretation of pollen chemical composition in response to environmental conditions. The study covered three grass species, Poa alpina, Anthoxanthum odoratum, and Festuca ovina. For each species, plants were grown from seeds of three populations with wide geographic and climate variation. Each individual plant was divided into four genetically identical clones which were grown in different controlled environments (high and low levels of temperature and nutrients). In total, 389 samples were measured using a high-throughput FTIR spectrometer. The biochemical fingerprints of pollen were species and population specific, and plastic in response to different environmental conditions. The response was most pronounced for temperature, influencing the levels of proteins, lipids, and carbohydrates in pollen of all species. Furthermore, there is considerable variation in plasticity of the chemical composition of pollen among species and populations. The use of high-throughput FTIR spectroscopy provides fast, cheap, and simple assessment of the chemical composition of pollen. In combination with controlled-condition growth experiments and multivariate analyses, FTIR spectroscopy opens up for studies of the adaptive role of pollen that until now has been difficult with available methodology. The approach can easily be extended to other species and environmental conditions and has the potential to significantly increase our understanding of plant male function.

Host and tissue variations overshadow the response of boreal moss-associated fungal communities to increased nitrogen load.

Human activity has more than doubled the amount of nitrogen entering the global nitrogen cycle, and the boreal forest biome is a nitrogen-limited ecosystem sensitive to nitrogen load perturbation. Although bryophyte-associated microbes contribute significantly to boreal forest ecosystem function, particularly in carbon and nitrogen cycling, little is known about their responses to anthropogenic global change. Amplicon pyrosequencing of the ITS2 region of rDNA was used to investigate how fungal communities associated with three bryophyte species responded to increased nitrogen loads in a long-term fertilization experiment in a boreal Picea abies forest in southern Norway. Overall, OTU richness, community composition and the relative abundance of specific ecological guilds were primarily influenced by host species identity and tissue type. Although not the primary factor affecting fungal communities, nitrogen addition did impact the abundance of specific guilds of fungi and the resulting overall community composition. Increased nitrogen loads decreased ectomycorrhizal abundance, with Amphinema, Cortinarius, Russula and Tylospora OTUs responding negatively to fertilization. Pathogen abundance increased with fertilization, particularly in the moss pathogen Eocronartium. Saprophytic fungi were both positively and negatively impacted by the nitrogen addition, indicating a complex community level response. The overshadowing of the effects of increased nitrogen loads by variation related to host and tissue type highlights the complexity of bryophyte-associated microbial communities and the intricate nature of their responses to anthropogenic global change.

Towards a global assessment of pyrogenic carbon from vegetation fires.

The production of pyrogenic carbon (PyC; a continuum of organic carbon (C) ranging from partially charred biomass and charcoal to soot) is a widely acknowledged C sink, with the latest estimates indicating that ~50% of the PyC produced by vegetation fires potentially sequesters C over centuries. Nevertheless, the quantitative importance of PyC in the global C balance remains contentious, and therefore, PyC is rarely considered in global C cycle and climate studies. Here we examine the robustness of existing evidence and identify the main research gaps in the production, fluxes and fate of PyC from vegetation fires. Much of the previous work on PyC production has focused on selected components of total PyC generated in vegetation fires, likely leading to underestimates. We suggest that global PyC production could be in the range of 116-385 Tg C yr(-1) , that is ~0.2-0.6% of the annual terrestrial net primary production. According to our estimations, atmospheric emissions of soot/black C might be a smaller fraction of total PyC (<2%) than previously reported. Research on the fate of PyC in the environment has mainly focused on its degradation pathways, and its accumulation and resilience either in situ (surface soils) or in ultimate sinks (marine sediments). Off-site transport, transformation and PyC storage in intermediate pools are often overlooked, which could explain the fate of a substantial fraction of the PyC mobilized annually. We propose new research directions addressing gaps in the global PyC cycle to fully understand the importance of the products of burning in global C cycle dynamics.

Mapping Above- and Below-Ground Carbon Pools in Boreal Forests: The Case for Airborne Lidar.

A large and growing body of evidence has demonstrated that airborne scanning light detection and ranging (lidar) systems can be an effective tool in measuring and monitoring above-ground forest tree biomass. However, the potential of lidar as an all-round tool for assisting in assessment of carbon (C) stocks in soil and non-tree vegetation components of the forest ecosystem has been given much less attention. Here we combine the use airborne small footprint scanning lidar with fine-scale spatial C data relating to vegetation and the soil surface to describe and contrast the size and spatial distribution of C pools within and among multilayered Norway spruce (Picea abies) stands. Predictor variables from lidar derived metrics delivered precise models of above- and below-ground tree C, which comprised the largest C pool in our study stands. We also found evidence that lidar canopy data correlated well with the variation in field layer C stock, consisting mainly of ericaceous dwarf shrubs and herbaceous plants. However, lidar metrics derived directly from understory echoes did not yield significant models. Furthermore, our results indicate that the variation in both the mosses and soil organic layer C stock plots appears less influenced by differences in stand structure properties than topographical gradients. By using topographical models from lidar ground returns we were able to establish a strong correlation between lidar data and the organic layer C stock at a stand level. Increasing the topographical resolution from plot averages (~2000 m2) towards individual grid cells (1 m2) did not yield consistent models. Our study demonstrates a connection between the size and distribution of different forest C pools and models derived from airborne lidar data, providing a foundation for future research concerning the use of lidar for assessing and monitoring boreal forest C.

Spatial variability of organic layer thickness and carbon stocks in mature boreal forest stands--implications and suggestions for sampling designs.

Accurate field measurements from inventories across fine spatial scales are critical to improve sampling designs and to increase the precision of forest C cycling modeling. By studying soils undisturbed from active forest management, this paper gives a unique insight in the naturally occurring variability of organic layer C and provides valuable references against which subsequent and future sampling schemes can be evaluated. We found that the organic layer C stocks displayed great short-range variability with spatial autocorrelation distances ranging from 0.86 up to 2.85 m. When spatial autocorrelations are known, we show that a minimum of 20 inventory samples separated by ∼5 m is needed to determine the organic layer C stock with a precision of ±0.5 kg C m(-2). Our data also demonstrates a strong relationship between the organic layer C stock and horizon thickness (R (2) ranging from 0.58 to 0.82). This relationship suggests that relatively inexpensive measurements of horizon thickness can supplement soil C sampling, by reducing the number of soil samples collected, or to enhance the spatial resolution of organic layer C mapping.

Temperature, precipitation and biotic interactions as determinants of tree seedling recruitment across the tree line ecotone.

Seedling recruitment is a critical life history stage for trees, and successful recruitment is tightly linked to both abiotic factors and biotic interactions. In order to better understand how tree species' distributions may change in response to anticipated climate change, more knowledge of the effects of complex climate and biotic interactions is needed. We conducted a seed-sowing experiment to investigate how temperature, precipitation and biotic interactions impact recruitment of Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings in southern Norway. Seeds were sown into intact vegetation and experimentally created gaps. To study the combined effects of temperature and precipitation, the experiment was replicated across 12 sites, spanning a natural climate gradient from boreal to alpine and from sub-continental to oceanic. Seedling emergence and survival were assessed 12 and 16 months after sowing, respectively, and above-ground biomass and height were determined at the end of the experiment. Interestingly, very few seedlings were detected in the boreal sites, and the highest number of seedlings emerged and established in the alpine sites, indicating that low temperature did not limit seedling recruitment. Site precipitation had an overall positive effect on seedling recruitment, especially at intermediate precipitation levels. Seedling emergence, establishment and biomass were higher in gap plots compared to intact vegetation at all temperature levels. These results suggest that biotic interactions in the form of competition may be more important than temperature as a limiting factor for tree seedling recruitment in the sub- and low-alpine zone of southern Norway.

Increase in carbon accumulation in a boreal peatland following a period of wetter climate and long-term decrease in nitrogen deposition.

Rates of peat growth and carbon (C) accumulation in a Sphagnum-dominated boreal peatland in south-east Norway were compared over two time periods each 17 yr long, that is, an earlier period from 1978 to 1995 and a recent period from 1995 to 2012. Our research was based on 109 peat cores. By using exactly the same study area and sampling protocols to obtain data for the two time periods, we were able to obtain a clear picture of the spatio-temporal patterns of peat accumulation. We show that peat growth and C accumulation were significantly higher in the recent than in the earlier time period. Interestingly, nitrogen (N) deposition was lower in the recent than in the earlier time period, while precipitation increased in the recent time period. Temperatures did not show any consistent trends over the time periods. Although our data do not allow assessment of the relative importance of declining N deposition vs increasing precipitation as drivers of peat accumulation, our results suggest that peatland C sequestration is not significantly inhibited by N pollution at current precipitation and N deposition levels.

Forestry impacts on the hidden fungal biodiversity associated with bryophytes.

Recent studies have revealed an unexpectedly high, cryptic diversity of fungi associated with boreal forest bryophytes. Forestry practices heavily influence the boreal forest and fundamentally transform the landscape. However, little is known about how bryophyte-associated fungal communities are affected by these large-scale habitat transformations. This study assesses to what degree bryophyte-associated fungal communities are structured across the forest successional stages created by current forestry practices. Shoots of Hylocomium splendens were collected in Picea abies dominated forests of different ages, and their associated fungal communities were surveyed by pyrosequencing of ITS2 amplicons. Although community richness, diversity and evenness were relatively stable across the forest types and all were consistently dominated by ascomycete taxa, there was a marked shift in fungal community composition between young and old forests. Numerous fungal operational taxonomic units (OTUs) showed distinct affinities for different forest ages. Spatial structure was also detected among the sites, suggesting that environmental gradients resulting from the topography of the study area and dispersal limitations may also significantly affect bryophyte-associated fungal community structure. This study confirms that Hylocomium splendens hosts an immense diversity of fungi and demonstrates that this community is structured in part by forest age, and as such is highly influenced by modern forestry practices.

Ancient carbon from a melting glacier gives high ¹4C age in living pioneer invertebrates.

Glaciers are retreating and predatory invertebrates rapidly colonize deglaciated, barren ground. The paradox of establishing predators before plants and herbivores has been explained by wind-driven input of invertebrate prey. Here we present an alternative explanation and a novel glacier foreland food web by showing that pioneer predators eat locally produced midges containing 21,000 years old ancient carbon released by the melting glacier. Ancient carbon was assimilated by aquatic midge larvae, and terrestrial adults achieved a radiocarbon age of 1040 years. Terrestrial spiders, harvestmen and beetles feeding on adult midges had radiocarbon ages of 340-1100 years. Water beetles assumed to eat midge larvae reached radiocarbon ages of 1100-1200 years. Because both aquatic and terrestrial pioneer communities use ancient carbon, the term "primary succession" is questionable in glacier forelands. If our "old" invertebrates had been collected as subfossils and radiocarbon dated, their age would have been overestimated by up to 1100 years.

Amplicon-pyrosequencing-based detection of compositional shifts in bryophyte-associated fungal communities along an elevation gradient.

Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low-alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.

Microsatellite markers for Hylocomium splendens (Hylocomiaceae).

PREMISE OF THE STUDY: The perennial feather moss Hylocomium splendens is one of the most widely distributed and common bryophytes in the Northern Hemisphere and has, because of its capacity to grow under a wide range of environmental conditions, been used as a biomonitor for atmospheric metal deposition in Europe. METHODS AND RESULTS: We present a multiplex approach for the analysis of 14 microsatellite markers tested on 194 H. splendens gametophytes. Ten of the markers are developed recently, and are presented for the first time in this paper, whereas four were previously developed but have not been used for population genetic investigations. CONCLUSIONS: The microsatellite markers reported here will provide a powerful tool for further research on population genetic structure in H. splendens.