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ISBN 978-951-42-9396-2 (Paperback)ISBN 978-951-42-9397-9 (PDF)ISSN 0355-3191 (Print)ISSN 1796-220X (Online)

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OULU 2011

A 571

Sari Hilli

CARBON FRACTIONS AND STOCKS IN ORGANIC LAYERS IN BOREAL FOREST SOILS—IMPACTS OF CLIMATIC AND NUTRITIONAL CONDITIONS

UNIVERSITY OF OULU,FACULTY OF SCIENCE, DEPARTMENT OF BIOLOGY;FINNISH FOREST RESEARCH INSTITUTE,NORTHERN REGIONAL UNIT

A 571

ACTA

Sari Hilli

A C T A U N I V E R S I T A T I S O U L U E N S I SA S c i e n t i a e R e r u m N a t u r a l i u m 5 7 1

SARI HILLI

CARBON FRACTIONS AND STOCKS IN ORGANIC LAYERS IN BOREAL FOREST SOILS—IMPACTS OF CLIMATIC AND NUTRITIONAL CONDITIONS

Academic dissertation to be presented with the assent ofthe Faculty of Science of the University of Oulu for publicdefence in Kuusamonsali (Auditorium YB210), Linnanmaa,on 6 May 2011, at 12 noon

UNIVERSITY OF OULU, OULU 2011

Copyright © 2011Acta Univ. Oul. A 571, 2011

Supervised byDoctor John DeromeDocent Annamari MarkkolaDoctor Sari Stark

Reviewed byProfessor Ingrid Kögel-KnabnerDoctor Gaius R. Shaver

ISBN 978-951-42-9396-2 (Paperback)ISBN 978-951-42-9397-9 (PDF)http://herkules.oulu.fi/isbn9789514293979/ISSN 0355-3191 (Printed)ISSN 1796-220X (Online)http://herkules.oulu.fi/issn03553191/

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2011

Hilli, Sari, Carbon fractions and stocks in organic layers in boreal forestsoils—impacts of climatic and nutritional conditions. University of Oulu, Faculty of Science, Department of Biology, P.O. Box 3000, FI-90014University of Oulu, Finland; Finnish Forest Research Institute, Northern Regional Unit, P.O.Box 16, Eteläranta 55, FI-96301 Rovaniemi, FinlandActa Univ. Oul. A 571, 2011Oulu, Finland

AbstractThe SOM in boreal forests contains non-living heterogeneous components resulting frommicrobial and chemical transformations of organic debris from plant litter. The major componentsin the plant biomass all decompose at different rates and therefore, contribute variably to the stablestorages of soil C. The aims of the current thesis were 1) to explore how climate, soil fertility andinitial litter quality affect the decomposition rate of litter, 2) to study how the different carbonfractions found in the plant litter relate to the quality and quantity of SOM in forest soils, 3) todetermine whether the recalcitrant fraction of litter is derived from lignin and other polyphenolsor from lipophilic compounds and carbohydrates, and 4) to determine whether the litter originatingfrom different plant growth forms affects SOM formation in a similar way. The study wasconducted in six north boreal and six south boreal study sites, half of which were mesic and halfwere sub-xeric. The overall initial litter quality and decomposition rate of carbon fractions did notdiffer between the two fertility levels and climate regimes. Litter with high initial water-solubleextractives (WSE) and nitrogen (N) decomposed at a faster rate than litter with lower initial WSEand N concentration irrespective of the soil fertility or climate conditions. Althoughdecomposition rate varies among litter types, decomposition rate cannot explain differences inSOM quality or quantity between the northern and southern boreal forests. The organic matteraccumulation and relative proportion of acid-insoluble residue (AIR) in SOM was higher in southboreal sites both in sub-xeric and mesic sites. Detailed characterization of the AIR fraction usingpyrolysis-GC demonstrated that in the litter layer the concentration of AIR contains lignin andother insoluble polyphenols, but in the F and H layers, lignin-derived and chemically modifiedpolyphenolics and decomposition products of resin acids. Although the concentration of the AIRfraction varies among litter types, its composition varied very little among the litter types, andbetween a sub-xeric and a mesic site. The results of this study suggest that the differences in plantcommunity structure and quantitative differences in the litter input by various growth forms hasmore impact on OM accumulation than decomposition conditions in coniferous soils.

Keywords: AIR, decomposition, lignin, lipophilic compounds, litter, organic matter,polyphenols, resin acids

Hilli, Sari, Ilmaston ja kasvillisuuden vaikutus hiilen fraktioihin boreaalistenmetsien maaperän orgaanisissa kerroksissa. Oulun yliopisto, Luonnontieteellinen tiedekunta, Biologian laitos, PL 3000, 90014 Oulunyliopisto; Metsäntutkimuslaitos, Pohjois-Suomen alueyksikkö, PL 16, Eteläranta 55, 96310RovaniemiActa Univ. Oul. A 571, 2011Oulu

TiivistelmäMetsämaan orgaaninen aines koostuu eriasteisesti hajonneesta karikkeesta sekä pitkälle maatu-neesta, hajottajien muokkaamasta humuksesta. Tutkimuksessa selvitettiin 1) kuinka ilmasto,maaperän ravinteisuus ja karikkeen kemialliset ominaisuudet vaikuttavat karikkeen hajoamisno-peuteen, 2) kuinka kasvien sisältämät erilaiset hiilifraktiot vaikuttavat maaperän orgaanisenaineen laatuun ja määrään, 3) millaiset hiiliyhdisteet muodostavat vaikeimmin hajoavan hiilif-raktion karikkeessa ja pidemmälle maatuneessa orgaanisessa aineessa ja 4) onko erilaisten kasvi-en vaikutus orgaanisen aineksen muodostumiseen samanlainen kuusikoissa ja männiköissä poh-jois- ja eteläboreaalisella havumetsävyöhykkeellä. Tutkimuksessa havaittiin, että karikkeen maa-tumisnopeudessa ei ollut eroja pohjois- ja eteläboreaalisella kasvuvyöhykkeellä eikä kuusikoi-den ja männiköiden väillä. Karike, joka sisälsi runsaasti vesiliukoisia yhdisteitä ja typpeä maa-tui nopeammin kuin vähän vesiliukoisia yhdisteitä tai typpeä sisältävä karike. Karikkeen maatu-misnopeus ei kuitenkaan kyennyt selittämään eroja pidemmälle maatuneen orgaanisen aineksenmäärässä ja laadussa pohjois- ja eteläboreaalisella kasvuvyöhykkeellä. Orgaanisen aineksenmäärä oli suurempi eteläboreaalisella kasvuvyöhykkeellä verrattuna pohjoisboreaaliseen. Lisäk-si vaikeasti hajoavia hiiliyhdisteitä (AIR-fraktio) kertyi eteläboreaaliseen metsämaahan enem-män kuin pohjoiseen. Karikekerroksessa ligniini ja polyfenolit muodostivat suuren osan AIR-fraktiosta. Maatumisen edetessä AIR-fraktion muodostavat lähinnä ligniinin hajoamisesta peräi-sin olevat ja muut modifioituneet polyfenolit sekä pihkahappojen hajoamistuotteet. Vaikka AIR-fraktion konsentraatio vaihteli kariketyypeittäin, sen kemiallinen koostumus oli hyvin samanlai-nen karikelajista riippumatta. Tutkimuksen perusteella vallitseva kasvillisuus ja sen tuottamankarikkeen määrä ja laatu vaikuttavat enemmän metsämaahan kertyvän orgaanisen aineen mää-rään kuin maatumisolosuhteet.

Asiasanat: AIR, karike, ligniini, maatuminen, orgaaninen aine, pihkahapot, polyfenolit,rasvat

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AcknowledgementsI owe my most grateful thanks to my supervisor the late John Derome. John offered me the opportunity to conduct this work in Metla and guided to me to the fi eld of soil science. I am happy that I had a possibility to work with an expert like John. John was an encouraging and a patient supervisor during all those years, when we worked together. John already knew, with his experience, that soil processes are complicated to investigate and no simple answers exist. However, he encourage me to continue with my work and allowed me to choose my interest in the fi eld of soil scinece.

I also want warmly thank my two other supervisors, Annamari Markkola and Sari Stark, who had very different, but important roles in my work. Annamari helped me with practical issues in University of Oulu and gave valuable comments to manuscripts. During writing process of this synthesis Annamari guided me with her knowledge and experience and encouraged me to follow that scientifi c path I have chosen. With Sari I started to work in John’s project in Metla. I am very grateful to Sari for all advice both practical and scientifi c I got during my work, as well as for cooperation, and friendship during all these years. We have had a great time with science, but also with dogs! Sari, if someone, you have shared all those feelings from frustrated and sadness to happy and success with me, many thanks of that.

Annika Smeds, Stefan Willför, Markku Reunanen and Matias Penttinen, your knowledge and help was more than important, thank you all for help. I am particularly grateful to Reijo Hautajärvi and also Pekka Välikangas, for organizing pretreatment of soil samples at Salla Research Unit and all those people who worked with me and my samples in Salla. Many thanks to Maija Salemaa, Leena Hamberg, Liisa Kulmala, Anneli Viherä-Aarnio, Petteri Muukkonen and Anna-Maija Kokkonen for their help at fi eld work. I also warmly thank Sirkka Aakkonen, Riitta Nielsen, Leena Seitamäki and Anri Rissanen for assistance in the laboratory work in Rovaniemi, Arja Ylinen in Parkano, and Raimo Pikkupeura for editing fi gures. I also thank all those not mentioned by name, who helped me with my work in fi eld and laboratory. Many thanks to Dr. Ingrid Kögel-Knabner and Dr. Gaius Shaver for reviewing this thesis and Aaron Bergdahl for checking and editing the English language. My PhD work was fi nancially supported by the Forest Focus pilot programme (EU, coordinated by John Derome at Metla), the Academyof Finland, the Ella and Georg Ehrnrooth Foundation, and the Faculty of Science University of Oulu.

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Finally, I warmly thank my friends, and my family, who have supported me during this process. Especially I thank my mate Anssi, who never complained, despite so much time I have spent with this work, and of course to my little assistant Siiri, a smooth hair fox terrier, who waitfully laid on and under working table waiting me to out.

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AbbreviationsSOM/OM soil organic matter, organic matterL Litter l ayerF Fermentation l ayerH Humus l ayerC CarbonN NitrogenC/N carbon nitrogen ratioNPE Non polar extractivesWSE Water soluble extractivesAS Acid soluble, fraction e.g. celluloseAIR Acid insoluble residue WEC Water extractable carbonWEN Water extractable nitrogenGC-MS Gas-chromatography mass spectrophotometry

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List of original articlesI Hilli S, Stark S & Derome J (2010) Litter decomposition rates in relation

to litter stocks in boreal coniferous forests along climatic and soil fertility gradients. Applied Soil Ecology 46: 200–208.

II Hilli S, Stark S & Derome J (2008) Carbon quality and stocks in organic horizons in boreal forest soils. Ecosystems 11: 270–282.

III Hilli S, Stark S & Derome J (2008) Qualitative and quantitative changes in water-extractable organic compounds in the organic horizon of boreal coniferous forests. Boreal Environment Research 13(supp.B): 107–119.

IV Stark S, Hilli S, Willför S, Smeds A, Reunanen M, Penttinen M & Hautajärvi R (2010) Composition of lipophilic compounds and carbohydrates in the accumulated plant litter and soil organic matter in boreal forests. Submitted manuscript.

V Hilli S, Stark S, Willför S, Smeds A, Reunanen M, & Hautajärvi R (2010) What is the composition of AIR? Chemical characterization of litter and soil in two south boreal forests using sequential fractionation and pyrolysis- GC-MS. Submitted manuscript.

Author’s contribution: The experimental design (I, II, III, IV, V) was planned by S. Hilli and J. Derome with contribution by S. Stark in paper I. S. Hilli organized the collection of samples for papers I–V and contributed to the sample collection together with the technical staff of Forest Research Unit of Finland (Metla). Sorting of samples (I–V) was planned and organized by S. Hilli and J. Derome. S. Hilli had the main responsibility for the tasks of organic fractionation and other chemical analyses in the laboratory (I–III), which were supervised by S. Stark. The chemical analyses of papers IV and V were planned by S. Willför and A. Smeds and executed by M. Penttinen and M. Reunanen. Interpretation of the detailed chemical analyses was done in collaboration with S. Willför and A. Smeds. Statistical analyses were executed by S. Hilli and S. Stark. S. Hilli had the main responsibility for data analysis in all papers (I–V) and writing of papers I–III and V, and had a major contribution in the writing of paper IV.

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ContentsAbstractTiivistelmäAcknowledgements 7Abbreviations 9List of original articles 11Contents 131 Introduction 15

1.1 Soil organic matter in boreal coniferous forests ..................................... 151.2 Impact of litter chemical quality to SOM accumulation ........................ 18

1.2.1 Water soluble-compounds ........................................................... 181.2.2 Cellulose, hemicelluloses and starch ........................................... 191.2.3 Lignin, condensed tannin ............................................................. 201.2.4 Soil lipids ..................................................................................... 21

1.3 The effect of boreal forest vegetation on decomposition and SOM s tocks ............................................................................................. 22

1.4 Impact of climate and soil fertility to decomposition of litter and OM accumulation .................................................................................. 24

2 Aims of the research 273 Material and Methods 31

3.1 Study sites ............................................................................................... 313.2 Litter decomposition experiment ............................................................ 313.3 Soil samples of organic layer ................................................................. 313.4. Litter and organic matter characterization by sequential fractionation ..... 32

3.4.1 Detailed characterization of the water soluble extractives (WSE), non-polar extractives (NPE), and acid soluble (AS) fractions ....................................................................................... 343.4.3 Detailed study of acid insoluble (AIR) fraction ........................... 34

3.5 Other chemical analyses ......................................................................... 34 3.6 Statistical analyses .................................................................................. 35

4 Results and Discussion 374.1 How does the chemical quality of litter differs between different stages of decomposition in boreal coniferous soils? .............................. 37

4.1.1 Water-soluble compounds (WSE fraction) .................................. 364.1.2 Extractable lipids (NPE fraction) ................................................. 384.1.3 Cellulose and hemicellulose (AS fraction) .................................. 39

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4.1.4 Acid insoluble residue (AIR) ....................................................... 404.1.5 A synthesis: How does the litter quality infl uence the SOM quality, and is recalcitrant carbon derived from lignin, other polyphenols, lipophilics, or carbohydrates? ....................... 42

4.2 How does the climate affect decomposition rate of organic fractions? ..... 434.3 How does the soil fertility affect decomposition of organic fractions? ..... 454.4 Determinants of soil carbon stocks in boreal forest soils ...................... 46

5 Conclusions and future perspectives 496 References 51Original articles 67

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1 Introduction1.1 Soil organic matter in boreal coniferous forests

Soil organic matter (SOM) originates mainly from leaf, wood and root litter and root exudates of plants and soil microbes. The SOM acts as an important reservoir of plant nutrients and carbon, and acts as energy and nutrient source for microbes and other decomposers (Berg 2000, Karhu et al. 2010). SOM has an important role in water retention in soils, as the water-holding capacity increases along with the quantity of SOM (Zhi-hua et al. 2003, Rajkai 2008). Stable soil moisture conditions have a generally positive effect on the biomass of the decomposer soil microorganisms (Donnelly 1990). The quality of SOM, especially nutrient concentrations and pH, affect plant species and microbial community composition (Pennanen 2001, van Oijen 2005) and plant regeneration (Ponge et al. 1998). Because of the nature of organic matter, SOM has a great ability to bind and stabilize compounds in soils (Lützlow et al. 2006). The SOM interacts with metal ions, oxides, hydroxides, mineral and organic compounds, including toxic pollutants such as polyaromatic carbon compounds and heavy metals, to form water-soluble and water-insoluble complexes (Monreal et al. 1997, Dube et al. 2001, Linnik 2003).

In boreal soils, climate affects the litter decomposition rate directly through temperature and moisture (Aerts 1997, Chen et al. 2000). Climate can also have an indirect effect on litter chemistry and decomposition rate through infl uences on plant community composition (Aerts 1997, Hobbie 2000). The litter decomposition process is slow in predominantly cold, wet and nutrient-limited environments (Berg et al. 1982, Hobbie 1996). This generally leads to accumulation of SOM on the soil surface. In northern ecosystems decomposition is also limited by the long winter, even though some microbes are active and litter decomposition occurs under the snow cover (Clein & Schimel 1995, Hobbie 2000, Grogan & Johansson 2005). Some studies suggest (e.g. Côuteaux et al. 1995) that in unfavorable weather conditions, such as areas at high Northern latitudes, climate may be a more important determinant of the decomposition rate at late stages than the quality of litter.

Due to the accumulated SOM, forest soils in the boreal zone are an important reservoir for global carbon (C) (Goodale et al. 2002). Thus, boreal SOM is an important component of the global carbon cycle. Understanding the mechanisms of C sequestration to SOM has attracted considerable attention in recent years. This is because of important questions concerning how organic C stocks will respond to a change in the global climate, which is expected to be the greatest in the northern boreal

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and sub-arctic and arctic regions (Vucetich et al. 1999). The temperature may increase by 1–2°C in summer and by 2–3°C in winter over the next 50 years (Carter et al. 2005). Longer and warmer growing seasons may increase primary production (Bergh et al. 2003), but it is suggested that decomposition rates may be enhanced to a greater extent than primary production (Kirschbaum 1995, 2000, Schimel et al. 1994). For predicting responses to changes in the environment, it is imperative to understand the processes and mechanisms that determine the decomposition and accumulation rates of SOM.

When fresh litter is deposited on the soil surface, it begins to undergo the processes of decomposition, which in time forms a natural decomposition continuum along the vertical soil gradient. During the decomposition of non-humifi ed organic materials (remains of plants, animals and microorganisms) in soil, a variable proportion of organic C (30–80%) is reverted to the atmosphere as CO2 (González-Pérez et al. 2004, Charro et al. 2010). This is a rapid mineralization process that usually takes place during the fi rst year of litter decomposition. The remaining, non-mineralized C undergoes a slower oxidation process and, after complex transformations, it either turns into microbial biomass or is stabilized in the form of humic substances (Stevenson 1994, González-Pérez et al. 2004). The proportion of the plant litter C which is transformed into stable organic matter is highly recalcitrant, accumulating as SOM (Lorenz et al. 2007, Kögel-Knabner et al. 2008). On average, 1–2% of plant residues become stabilized as humifi ed SOM for signifi cant periods of time (Schlesinger 1990). The SOM composition ranges from non-living heterogeneous components resulting from microbial and chemical transformations of organic debris (Stevenson 1982) to more decomposed material in the humus of surface soils and mineral soil (Qualls et al. 2003, Lorenz et al. 2007).

The different layers in the organic horizon are distinguished by the degree of decomposition. Litter layer (L) is limited to the accumulation of organic debris (leaves, twigs, straw, moss, etc.) in the uppermost part of the organic-rich soil layer. The origin of plant material is recognizable in the L layer, whereas an underlying fermentation (F) layer represents a biofermented organic horizon with intense biological activity by bacteria and fungi. Finally, in the lowermost brown humus (H) layer the origin of litter or plant structure is no longer distinguishable (The Canadian System of Soil Classifi cation 1998). Coniferous litter decomposes over a long period and new litter is deposited on top of the older litter before the latter is depleted of readily available C (Berg & McClaugherty, 2003). The humifi cation of organic matter increases in the sequence L < F < H (Rumpel et al. 2006). SOM and C pools of the L, F and H layers are not stable, but carbon compound transformations between soil layers characterize the decomposition stage (Zech et al. 1997, Karhu et al. 2010).

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Recalcitrant organic material, generically referred to as humus or humic substances is often described as “a series of relatively high molecular weight, brown to black colored substances, formed by a secondary synthesis reaction“ (Konova 1966, Stevenson 1982). Recalcitrant SOM also contains nonhumic substances, e.g., amino acids, lipids, carbohydrates (Magdoff et al. 1996) and root exudates (Zech et al. 1997). With the aid of advanced molecular techniques, a new view of humic acids describes them as dynamic supramolecular associations (Kleber et al. 2007, Piccolo 2001) of relatively low molecular weight components. These components include recognizable but often partially-oxidized biomolecules (Nelson & Baldock 2005, Schnitzer 2000).

The chemical composition of SOM, especially the proportion of “labile” versus “recalcitrant” carbon, is considered important for the stabilization of SOM in the soil organic horizon. However, contrasting views exist on the stabilization mechanisms of SOM (Ekschmitt et al. 2005, Lützlow et al. 2006, Marschner et al. 2008). Based on earlier studies, lignin and its transformation products, as well as polyphenols and derived polymers from lower plants and microorganisms, are important building materials of recalcitrant SOM by providing aromatic building blocks of high physicochemical stability (Stevenson 1994). The formation of humic acids from lignin is due to simultaneous degradation of the side chain of lignin and polymerization of degradation products (Flaig 1964). Conventional explanations for the formation of stable organic compounds assumed them to be stable due to the highly aromatic structure of humic acids. This concept has often been questioned in the past few decades (Hedges et al. 2000, Kögel-Knabner et al. 1992a,1992b, Qualls et al. 2003, Marschner et al. 2008).

A more recent investigation shows that “recalcitrant carbon” also has a large potential to degrade (Lützlow et al. 2006, Kleber 2010, Karhu 2010). According to this view, the stability of OM is not related to the inherent recalcitrance of the components; rather, the functional groups create an environment where enzymes are no longer effective in degrading the substrate. Of particular importance to soil organic carbon (SOC) sequestration is the recognition that simple relatively-fresh biomolecules can directly contribute to this recalcitrant SOM pool. Thus, there does not necessarily have to be a long slow aging process to produce stable humus, and the old organic matter may contain easily metabolizable organic molecules with low thermodynamic stability (Qualls et al. 2003, Kleber et al. 2010). This view is in line with fi ndings indicating that recycling of plant C by microbes plays an important role in the formation of SOM (Amelung et al. 2002, Karhu et al. 2010).

The identity of the carbon compounds that accumulate in the SOM is methodologically often assessed by analyzing the chemical composition of

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decomposing litter and SOM in various stages of decomposition. For example, characterization of the litters and SOM in the different layers from the L to the F layer, and further to the H layer provides important insight into the role of different organic substances in different phases in the decomposition process and its effect on the accumulation of recalcitrant SOM (Berg 2000). Investigation of the chemical changes along litter and SOM decomposition in different vegetation types, ecosystem productivity levels, and the climatic zones gives information about the role of these factors in SOM decomposition and accumulation rates.

1.2 Impact of litter chemical quality on SOM accumulation

The major components in the plant biomass (e.g. carbohydrates, cellulose and hemicellulose, lipophilic compounds, lignin and other polyphenols) all decompose at different rates and therefore, may contribute variably to the storage of soil C. Generally, high concentrations of water-soluble compounds may indicate rapid decomposability, whereas high concentrations of insoluble compounds, such as the acid insoluble residue (AIR) fraction in litter, may generally indicate high resistance to decomposition (Preston et al. 1997, Shaver et al. 2006). Also a large concentration of carbohydrates with a low concentration of extractives indicates rapid accumulation of resistant biopolymers (Almendros et al. 2000). The relative role of aliphatic compounds, such as cellulose and other carbohydrates, and phenolic compounds have been proposed to contribute recalcitrant compounds to SOM (de Leeuw & Largeau 1993, de Leeuw et al. 2006, Kögel-Knabner 2002).

1.2.1 Water-soluble compounds

Soluble organic compounds consist of sugars, low molecular weight phenolics (e.g. hydrolysable tannins, fl avonoids), hydrocarbons and glycerides. These compounds are generally rapidly degraded or lost from the litter through dissolution and leaching during the fi rst year of decomposition (Berg et al. 1982). The water-soluble organic compounds in decomposing organic matter are considered to form the most active soil carbon pool (Ibrahima et al. 1995, Kalbitz & Kaiser 2007). Therefore, despite constituting a relatively small fraction of the total amount of organic matter in the soil, these compounds have an important effect on the degradation and mineralization rates of organic matter (Miltner & Zech 1998, Gallet & Keller 1999). The concentration and the proportion of water-soluble compounds in fresh and senescent plant biomass and litter differ within and among tree and understory

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vascular plant and moss species (Hobbie 1996, Gallet et al. 1999, Girisha et al. 2003, Wardle et al. 2003, Don & Kalbitz 2005).

Soil carbohydrates are a mixture of complex polysaccharides composed of monosaccharides. Five monosaccharides, including glucose, galactose, mannose, arabinose, and xylose, are the most common compounds among the total hydrolyzable carbohydrates in plants and plant litter (Chesire 1977, Anderson & Hetherington 1999). The hydrolysable carbohydrates are mainly derived from plant hemicelluloses, pectin and soil microbes (Evans et al. 2001, Kögel-Knabner 2002). The highest sugar concentration of forest soils were earlier found in litter and the F layer with fairly intact plant material (Rumpel & Dignac 2006). The saccharides released from decomposing organic litter are a source of C for soil microbes and easily incorporated into microbial tissues, mineralized or polymerized or adsorbed into soil (Stevenson 1994).

Phenolics comprise a large group of secondary metabolites with a wide range of chemical properties, from the low molecular weight phenolic acids to the high molecular weight condensed tannins. The fate of these compounds in soil may vary considerably among the different types of phenolic compounds (Hättenschwiler & Vitousek, 2000, Balaria et al. 2009, Coulis et al. 2009). Vegetation is the main source of phenolic compounds in soils, although microbial synthesis and transformation also occur (Swift et al. 1979). A proportion of phenolic compounds, such as hydrolyzable tannins, is soluble and usually decreases rapidly during the decomposition process (Preston et al. 1997, Coulis et al. 2009). The water-extractable phenols in the H layer most probably consist of humifi ed rather than of plant-derived substances (Fröberg et al. 2003, Michalzik et al. 2003) and probably constitute the most stable water-soluble carbon pool in soils (Kalbiz & Kaiser 2007).

1.2.2 Cellulose, hemicelluloses and starch

Cellulose, hemicelluloses and starch in organic material is often analyzed as the acid-soluble fraction (AS) (Hobbie 1996). The AS content of litter differs within and among tree and understory vascular plant and moss species (Hobbie 1996, Gallet et al. 1999, Girisha et al. 2003, Tian et al. 2000, Don & Kalbitz 2005). Compared to water-soluble sugars and other extractives, the AS fraction decomposes slowly (Tian et al. 2000, Girisha et al. 2003). The simple sugars are polymerized in the plants to form long polysaccharide chains which are intertwined to make up the plant fi bers. Cellulose is a linear polymer composed of glucose units, which is closely associated with hemicelluloses and in the woody cell walls to lignin (Kögel-Knabner 2002).

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Cellulose thus occurs in high concentrations in soil organic materials. Pentoses, xylose and arabinose, and hexoses, glucose, mannose, and galactose are the major components of hemicelluloses in plants. In coniferous wood hemicellulose sugars are xylans (formed by xylose, 4-O-methylglucuronic acid), mannans (mannose, glucose, galactose and acetyl groups) and galactans (galactose, arabinose) (Fengel & Wegener 1984). Of hemicellulose sugars arabinose and galactose components seem to be most easily degraded, followed by mannose and xylose (Curling et al. 2001). In general hemicellulose is decomposed at a greater rate than cellulose (Kögel-Knabner 2002).

1.2.3 Lignin, condensed tannin

Lignin is found in all vascular plants, a major fraction being distributed throughout the secondary walls of woody cells and also in the middle lamella between the secondary cell walls (Eriksson et al. 1990). The term ‘lignin’ is commonly used to signify the fraction obtained by proximate analysis, e.g. Klason lignin’ or acid-insoluble residue was the term used to describe the remaining substance after sulphuric acid hydrolysis (Preston et al. 1997). When analyzed in litter or SOM, this fraction contains not only ‘true’ lignin but also tannins and cutin (Preston et al. 1997, Almendros 2000) and may also include humic substances and fungal compounds (Osono & Takeda 2006). Klason lignin content of coniferous woods may vary from 26.8–32.1% depending on species (Sjöström 1993).

Lignin is synthesized by heterogenous composition of cinnamyl, coniferyl, sinapyl and p-coumaryl alcohol monomers and different types of bonds (Kögel-Knabner 2002). Guaiacyl lignin is mainly found in softwoods (24–33% of dry biomass), guaiacyl-syringyl lignin (16–25%) in hardwoods and grasses contain guaiacyl-syringyl-p-hydroxyphenol lignin (< 20%) (Kapoor et al. 2005). Lignin in pine trees is almost exclusively composed of coniferyl alcohol units (Nierop et al. 2001) producing guaiacyl lignin, which is more recalcitrant than other natural lignins (Faix et al. 1985). Condensed tannins and lignin are the most abundant polyphenols in woody plants, but they are usually absent from herbaceous plants (reviewed by Hättenschwiler & Vitousek 2000, Lorenz et al. 2000). Condensed tannins are found in high concentrations in coniferous wood bark (Kögel-Knabner 2002, Hernes & Hedges 2004). High molecular weight phenolics in soils may consist of derivates of lignin degradation products, fl avonoids, tannins, carbohydrate and protein complexes (Smolander et al. 2005). Polyphenols are relatively resistant to microbial degradation and they can affect soil nutrient dynamics by forming complexes with

21

proteins, and in this way, conserve sequestered carbon (Hättenschwiler & Vitousek 2000, Kögel-Knabner 2002, Castells et al. 2004).

The appearance of guiaiacyl lignin monomers and oxidized forms of lignin monomers in litter and soil samples are evidence of biodegradation of guaiacyl lignin (Dijkstra et al. 1998, Preston et al. 1994). Although previously lignin was considered to be the main substance to regulate overall decomposition rates (Berg 2000), more recent studies indicate that lignin is decomposed rather rapidly and does not accumulate in the SOM to a great extent (Kiem & Kögel-Knabner 2003). However, there is some evidence showing that degradation products of lignin are found in coniferous soils especially in cooler climate (Otto & Simpson 2006).

1.2.4 Soil lipids

Lipids are, by classical defi nition, organic compounds insoluble in water, but soluble in common organic solvents (Kögel-Knabner 2002). The lipids in the SOM originate from plants, animals, or decomposer microorganisms, i.e. fungi, bacteria and meso- and micro-fauna (Bull et al. 2000). The main source of lipids in soils is usually vegetation (Oades 1993, van Bergen et al. 1997). The microbial biomass in boreal forest soils is largest at the organic layer (Fritze et al. 2000), thus also a high proportion of fungal and bacterial lipids are expected. In aerobic soils, lipids may account for 2 to 6% of soil humus (Dinel 1990, Stevenson 1994), showing a tendency to accumulate in the soil organic horizon (Naafs et al. 2004, Kainulainen & Holopainen 2002, Kanerva et al. 2008).

Free lipids in soils represent a diverse group of hydrophobic substances ranging from simple compounds such as fatty acids, to more complex substances as sterols, terpenes, polynuclear hydrocarbons, chlorophylls, fats, waxes, and resins (Gonza´lez-Pe´rez et al. 2004). Although the solvent-extractable lipids usually comprise less than 10% of the total OM, they provide useful information about the source and overall degradation of OM (Farella 2001). It has been suggested that some of the extractable lipid compounds can be easily mineralized in the soil (Almendros et al. 1996, van Bergen et al. 1997, Naafs et al. 2004). A decrease in steroids and terpenoids was observed in previous studies, when lipids from forest soil litter layers or leaf extracts were compared with those from the underlying A horizon (Jambu et al. 1993, van Bergen et al. 1997, Kanerva et al. 2008). In addition to being present as free extractable compounds, lipids are part of complex organic structures such as biopolyesters (Zelles et al. 1992, Stevenson 1994, Kögel-Knabner 2002, Winkler et al. 2005).

22

Cutin and suberin are believed to be the major sources for the detected lipids in stable SOM as fatty acids and alcohols with varying structures and chain length (Kögel-Knabner 2002, Winkler et al. 2005). Cutin is a major lipid found in plants, whereas suberin is abundant in belowground parts and is also found in cork tissues or bark (Bernards 2002, Kögel-Knabner 2002). Moucawi et al. (1981) have shown that enzymatic hydrolysis of both the waxes and the fats occur simultaneously and Otto and Simpson (2005) that abundance of wax lipids in plant material and soils decreased at higher rates than the steroids and terpenoids. However the lipids trapped within complex organic structures are non-extractable (Preston 1997, Kögel-Knabner 2000), and thus should be left over as AIR fraction in a proximate analysis like lignin derivates.

1.3 The effect of boreal forest vegetation on decomposition and SOM stocks

Boreal forests have a characteristic vegetation structure, consisting of a tree layer and understory of low-growing woody ericaceous shrubs (dwarf shrubs) and, frequently, mosses and lichens (Nilsson & Wardle 2005, Salemaa & Hamberg 2007). In north-western Europe, the dominant tree species are Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). The litter produced by Norway spruce has a higher N content than litter from Scots pine (Johansson 1995, Wardle et al. 2003, Ukonmaanaho et al. 2008), but lower contents of cellulose and galactan (Johansson 1995).

In boreal coniferous forests, a signifi cant proportion of net annual primary production originates from the ground vegetation (Kolari et al. 2006). The main components of understory vegetation in northern boreal forests are ericaceous dwarf shrubs in the fi eld layer and feather mosses and lichens in the ground layer (Salemaa & Hamberg 2007). Bilberry (Vacciniun myrtillus) and lingonberry (V. vitis-idaea) constitute the most common understory dwarf shrub species in Finland, although in nutrient-poor sites Calluna vulgaris and Empetrum nigrum dominate (Salemaa et al. 2008). V. myrtillus and V. vitis-idea contain high concentrations of phenolics (Wardle 2003), and the concentrations of both phenolics and nutrients are higher than in forest trees or other Ericaceous dwarf shrubs in boreal forests (Johansson 1993, Gallet & Lebreton 1995, Wardle et al. 2003). Boreal forests are characterized by a thick moss layer dominated by feather moss (Pleurozium schreberi) and stair-step moss (Hylocomium splendens). Lichens are common on nutrient-poor sites consisting mostly of Cladina or Cladonia species (Nilsson &

23

Wardle 2005, Salemaa et al. 2008). The moss layer is usually thicker in Norway spruce forests than in forests dominated by other tree species, such as Scots pine (Augusto et al. 2003), correlating positively with the water-holding capacity of the soil (Preston et al. 2006). The concentration of AIR in the mosses P. schreberi and H. splendens is very similar to that of the litter of P. sylvestris and P. abies and some dwarf shrubs, however the concentrations of water-soluble and total phenolics content differ (Wardle et al. 2003, Kanerva et al. 2008).

The plant growth rates, density of the tree canopy, and characteristics of the ground vegetation differ greatly in the different latitudinal zones of boreal forests (Vucetich et al. 1999, Ukonmaanaho 2008, Salemaa et al. 2008). The abundance of herbs and grasses increases from the north to the south and towards more fertile sites (Salemaa et al. 2008). Because different plant species have a variable composition of organic compound abundance, they also have very variable effects on the decomposition and carbon cycling rates (McTiernan et al. 2003, Fierer et al. 2001), and as well as soil carbon storage (Almendros et al. 2000, Hobbie et al. 2000). The ground vegetation of nutrient-poor, sub-xeric boreal forests primarily consists of evergreen dwarf shrubs that form phenolic rich, slowly decomposable litter (Gallet et al. 1999), but in nutrient-rich, mesic forests the decomposition rates are higher due to, e.g., lower concentrations of phenolic compounds in the plant litter (Flanagan & Van Cleve 1983). Feather mosses produce litter that decomposes slowly, and it has been shown that the rate of mass loss and N release from this litter is usually slower than that of the trees and dwarf shrubs with which they co-exist (Wardle et al. 2003). This often results in a thick layer of moss litter forming below the live moss portions and above the humus surface.

Tree species composition affects SOM directly through the differences in the litter chemistry, and indirectly through the effects on the understory ground vegetation and the microbial community composition (Priha et al. 1999, 2001, Fierer et al. 2001, Augusto 2003, Suominen et al. 2003, Grayston & Prescott 2005, Smolander et al. 2005). Although the total phenolic concentration in the H layer may be higher under birch (Betula sp.) than Norway spruce, the concentration of condensed tannins is higher under spruce than birch (Suominen et al. 2003, Smolander et al. 2005). Bonifacio et al. (2008) have shown that the organic matter accumulation and quality of humic substances in coniferous forest soils differs under different ground vegetation species while there are no differences in the humifi cation process. Changes in the plant species composition may have important consequences on SOM accumulation. For example, increasing abundance of V. myrtillus, which has high litter quality and nutrient concentrations, may reduce the

24

SOM stock, if it replaces mosses in the ground vegetation (Johannson 1993, Frank & Ponge 2002). However, some other ericaceous species produce litter with a high content of aromatic and aliphatic acids, tannins and stable carbohydrates, and in this way contribute signifi cantly to accumulation of SOM (Fjalal & Read 1983, Almendros et al. 2000) in e.g. Calluna soils (Fjalal & Read 1983).

1.4 Impact of climate and soil fertility to decomposition of litter and OM accumulation

Although the chemical quality of litter is an important determinant of its decomposition rate (Hobbie 1996), the conditions regulating decomposition processes also infl uence both the decomposition rate and the quality of the litter residues that accumulate in the organic layer of the soil (Berg & Meentemeyer 2001, Vucetich et al. 2000, Sariyildiz & Anderson 2003, McTiernan et al. 2003, Wardle et al. 2003, Kirschbaum 2006). Decomposition rate of plant litter is related to temperature, and a temperature increase of approximately 5 °C may double the decomposition rate (Hobbie et al. 2000). In addition microbial community structure may affect the temperature dependence of litter and SOM decomposition (Bradford et al. 2008, Vanhala et al. 2008, Karhu et al. 2010).

At present, two contrasting views exist on the role of chemical quality interacting with the impact of climate and temperature on the decomposition rates of litter and SOM. According to one view, in warm and moist conditions, soluble substrates may be decomposed more rapidly than in cool conditions. Therefore, the accumulation of recalcitrant substances (i.e. AIR fraction) is higher in a warm than in a cool climate (Berg et al. 1993, McTiernan et al. 2003). Another line of evidence suggests that the decomposition rates of recalcitrant litter and SOM respond more strongly to increased temperatures than the soluble components (Fierer et al. 2005, Hartley & Ineson 2008, Vanhala et al. 2008). Therefore, this view predicts that the temperature-dependence of decomposition increases with litter and SOM recalcitrance (Kirschbaum 1995, Bosatta & Ågren 1999). This is supported by some studies on leaf litter decomposition and organic soils (Hartley & Ineson 2008, Karhu et al. 2010). Furthermore, the effects of temperature may vary according to the temperature range. Some studies suggest that the temperature sensitivity of SOM decomposition increases towards lower temperatures (Kirschbaum 2000, 2006) and others that the SOM mineralization rate is equally dependent on temperature (Vanhala et al. 2008). Also the adaptation of the microbial community to use certain types of carbon sources infl uences SOM (Vanhala et al. 2008,

25

Merilä et al. 2010). Moisture or precipitation may also have varying effects on decomposition. In dry regions, precipitation is the most important limiting factor of decomposition (Fioretto et al. 1998) and outweighs the importance of temperature when comparing areas with the same mean annual temperature (Prescott et al. 2004a). High precipitation may enhance the decomposition of cellulose, but retard the decomposition of lignin and its constituents (McTiernan et al. 2003).

Similar to the views on the role of temperature, there are different views on the role of nitrogen (N) in the decomposition. The classic work by Berg and Staaf (1981) demonstrated the importance of litter C/N ratio on the litter decomposition rates. They hypothesized that in the initial stages of decomposition, N limits the decomposition rate, and N often accumulates in litter at an early stage of decomposition (Berg & Staaf 1981, Chadwick 1998, Moore 2006). The higher initial N content or addition N-rich fertilizer, however, has a negative effect on decomposition at later stages of decomposition, especially in the case of lignin-rich organic matter (Fog 1988, Berg & Ekbohm 1991, Coûteaux et al. 1995, Berg & Meentemeyer 2002, Berg & McClaugherty 2003, Hobbie 2008). High N content of organic matter stimulates the growth of micro-organisms capable of using labile C compounds as an energy source, but limits growth of fungi capable of degrading lignin (Swift et al. 1979, Coûteaux et al. 1991, Carreiro et al. 2000). In this way, the higher N content of litter limits degradation of lignin and leads to an increase of recalcitrance of soil organic matter (Ågren et al. 2001, Michel & Matzner 2002, Berg & Meentemeyer 2002, Berg & McClaugherty 2003). The idea that addition of N to litter decreases lignin degradation or increases recalcitrance of SOM is questioned later (Sjöberg et al. 2004, Allison et al. 2009), and the mechanisms for a negative effect of N additions on microbial activities in boreal forests are not entirely understood (Sjöberg et al. 2004). However, laboratory studies suggest that the initial lignin and N content of litter affects the capacity of fungi to use litter as an energy source and thus affects the decomposition rate of litter (Allison et al. 2009, Song et al. 2010).

Leaf litter decomposition and the mineralization of C on the forest fl oor is usually faster in more nutrient-rich conditions (Gosz 1973, Côté et al. 2000). However, N-rich litter or fertilized litter do not always decompose at a faster rate than unfertilized or control litter (Prescott 1995, Chen et al. 2004). Therefore, the ratio of easily soluble carbon fractions to the recalcitrant fractions may be a more important determinant of the decomposition rate than soil N availability (Murphy 1998, Sariyildiz & Andersson 2003). Although the decomposition rate of different litter types also varies among types of litter (Wardle et al. 2003), it is diffi cult

26

to assess to what extent the differences in the decomposition rates among plant species can be explained by the carbon quality and to what extent by the nutrient concentrations of litter. Prescott (2005) has suggested that due to use of correlation analyses, the most important predictor of decomposition rate in each case appears to be the factor with the greatest range or variance.

27

2 Aims of the researchIn this study I explored how the quality and amount of organic matter changes along the decomposition continuum in the organic layer under two different climate regimes and site fertility levels (I, II, III). Since the decomposition process is a continuum from freshly senescent litter to humus, I determined the proportion of soluble fractions (nonpolar, NPE, and water extractable, WSE) in relation to recalcitrant organic fractions (acid soluble, AS, and acid insoluble residue, AIR) in senescent litter, litter layer (L), fermentation layer (F) and humus layer (H). Fresh litter chemistry was studied to reveal differences in the chemical quality of litter between mesic and sub-xeric sites under different climate regimes. More detailed study of organic fractions and unfractionated samples gave information about the compounds that are dominant in each stage of decomposition and are preserved in the SOM (III, IV, V). I quantitatively analyzed the amount of main litter types in the L layer and the amount of SOM stored in the F and H layers, and therefore I am able to discuss the importance of tree, dwarf shrub and moss litter as a source of SOM (I, III). I also analyzed the decomposition rate of needle, V. myrtillus and moss litter and decomposition of different organic fractions, and in this way I discuss the importance of litter decomposition rates in the northern and southern boreal climate regimes and at two different stage of soil fertility (I) in relation to SOM accumulation.

The aims of this thesis were to answer the following questions:

1. How does the chemical quality of litter change along the different stages of decomposition in boreal coniferous soils?

As soluble and easily-decomposable substances generally decompose at greater rates than recalcitrant substances, the concentrations of soluble compounds gradually decrease and those of insoluble ones increase (Girisha et al. 2003, Berg 2000, McTiernan et al. 2003, Shaver et al. 2006). The proportion of AIR fraction or Klason lignin analyzed by proximate analysis increases (Almendros 2000, Tian 2000). Consequently, decomposing plant litter of very different plant origins should gradually become similar, and the heterogeneous plant material be converted into relatively homogeneous humus material (Melillo et al. 1989). I tested the following predictions:

28

1. The concentration of water-soluble compounds and total WSE fraction should decrease and that of AIR increase from fresh litter to the H layer (I, II).

2. The chemical quality of fresh litter (I) and accumulated litter in the L layer (I, II, III) should differ more by the site type and the climatic zone than the more decomposed material in the F and the H layers (II).

2. How does the litter quality affect the quality and quantity of SOM? Is the recalcitrant fraction of litter derived from lignin and other polyphenols or from lipophilic compounds and carbohydrates?

Some authors have suggested that the infl uence of substrate quality may affect decomposition and SOM formation more or, alternatively, be as important as temperature in boreal ecosystems (Flanagan & Cleve 1983, Hobbie et al. 2000). According to the traditional view, lignin and other aromatic polymers, like phenolic compounds, form the most recalcitrant fraction of SOM (Melillo et al. 1989, Berg 2000), but recent studies have demonstrated, that especially alkyl C compounds (Almendros et al. 2000, Lorenz et al. 2000, Kögel-Knabner 2002, Sjöberg et al. 2004), and other aliphatics such as cellulose and other carbohydrates produce recalcitrant compounds in SOM (de Leeuw & Largeau 1993, Nierop & Buurman 2001, Kögel-Knabner 2002, Buurman et al. 2005, de Leeuw et al. 2006). Based on previous studies, I tested the following prediction:

3. During the litter decomposition process from L to H layers the relative proportion of lignin decreases and the carbohydrate-based compounds and lipophilic compounds increase.

3. How does the climate and soil fertility affect decomposition and SOM quality and quantity?

The decomposition of different litter types is generally higher in warmer conditions and more nutrient rich sites (Berg et al. 1993, Coûteaux et al. 1998, McTiernan et al. 2003), but increasing N availability through fertilization or more N-rich litter does not always increase litter decomposition (Prescott et al. 2004). Soil OM that has developed under different climatic conditions may have varying chemical qualities and levels of resistance to decomposition (Berg et al. 1993). Climatic transects in litter decomposition experiments have demonstrated that, in warm and moist conditions accumulation of recalcitrant substances is higher than in cool conditions (Berg et al. 1993, Coûteaux et al. 1998, McTiernan et al. 2003). However, it is not

29

known to what extent the differences in the vegetation between the north and south boreal forests infl uence the litter C stocks produced by trees, understory dwarf shrubs and mosses, and to which extent these differences infl uence the quality and quantity of litter C stocks. I predicted that:

4. Because soluble compounds usually decrease at a faster rate in warmer conditions, the WSE fraction should decrease and the AIR increase at a faster rate at the south boreal than the north boreal sites, leaving a higher AIR concentration in the litter (I) and a higher proportion of AIR in the SOM in the south (II).

5. If nutrient availability limits decomposition, the overall decomposition rate of different litter material should be faster in mesic than in sub-xeric sites in both climate regimes (I). However, if the initial N concentration of the litter material is more important than the site fertility or climate, the litter with the highest N concentration should decompose at a faster rate than N-poor litter despite soil fertility or climate regime (I).

6. Although the decomposition rates of each litter type should be lower in the north than south boreal forests, differences in the ground vegetation between the north and the south boreal forests are also important determinants of the quality and quantity of SOM stocks in the L layer (I).

30

31

3 Material and Methods3.1 Study sites

In order to study the effects of soil fertility and climate I chose three Scots pine-dominated sub-xeric forest sites and three Norway spruce-dominated mesic forest sites in the north and south boreal climatic zone. The sub-xeric sites represented a low productive Empetrum-Myrtillus type in the north and Vaccinium type in the south. Mesic sites represented higher productivity, Hylocomium-Myrtillus type in the north and Oxalis-Myrtillus and Myrtillus type in the south (Salemaa et al. 2008). In 2003 during the growing season, the temperature sum in the south was 1328 and 813 in the north (Derome et al. 2007), and the mean annual temperature 3.1–4.1 °C in the south and -1 to 0 °C in the north boreal sites (Finnish Meteorological Institute). The mean annual temperature difference between north and south was 4.5 °C, which is within the range of warming scenarios (2.4–7.4 °C) for Finland (Jylhä et al. 2004).

3.2 Litter decomposition experiment

Decomposition rates in the south and north boreal and sub-xeric and mesic sites were analyzed in the fi eld with a litterbag experiment established in 2005. The investigated materials were 1) bilberry (Vaccinium myrtillus) leaves, 2) needle (Norway spruce, Scots pine) litter, and 3) moss litter. Bilberry leaves were sampled after 2 months, 6 months, and then from 1–3 years. Needle litterbags were sampled after 6 months, and then from 1–4 years. Moss litter samples were collected after 2 months, 6 months and then from 1–4 years.

3.3 Soil samples of organic layer

Natural soil profi les allow following of long-term changes in the relative proportions of the organic fractions during the process of decomposition, because each soil horizon represents a different stage of decomposition (Zech et al. 1992). For this reason, organic layer samples were taken in 2002 and 2003, starting in mid July and ending in mid August when the current year litter had still not fallen (II, III, IV, V). The intact soil samples (30 x 30 cm) of the forest fl oor were removed at regular intervals along four sampling lines at the edge of each square plot, resulting in 28 samples for each of the 12 plots, and a total of 336 samples. The total area of the squares on each plot was 2.52 m2. The samples contained the whole organic layer

32

(L, F and H) and all the living ground vegetation (including mosses, dwarf shrubs, etc.). The L layer was separated into the following fractions: needles, coarse tree litter, dwarf shrub litter, the dead parts of mosses and lichens, and herb and grass litter (IV), and dry weight was analyzed separately. The soil layers in the organic horizon were separated on the basis of their stage of humifi cation. The F layer consisted of brown or dark brown fragments of dead mosses and partly decomposed litter from the trees and ground vegetation. The H layer consisted of the most decomposed material, with only a few coarse pieces of tree litter. All visible roots and rhizomes were removed from the F and H layers, because most of them were alive and were therefore considered to be underground parts of living plants (unpublished data). The term stocks (g/m2) was used in this study to express the quantity of organic matter accumulated in the different layers in the organic horizons (L, F and H) (II). The litter fractions of the L layer were weighed separately (I), and the total L layer obtained as the sum of the individual litter components of the L layer (II, III).

3.4 Litter and organic matter characterization by sequential fractionation

In order to understand the ecology of accumulating C substrates, I characterized the soil organic matter (Table 1) according to Ryan et al. (1990) into the following fractions: nonpolar extractives, chloroform soluble (NPE, waxes, fatty acids, oils), water-soluble extractives (WSE, e.g. sugars and phenolics), acid-soluble fraction (AS, e.g. cellulose), and acid-insoluble residue (AIR) (lignin, tannin cutin (Ryan et al. 1990, Preston et al. 1997).

33

Tabl

e 1.

Sum

mar

y of

fi el

d sa

mpl

es a

nd c

hem

ical

ana

lyse

s in

the

pres

ent s

tudy

.

Dat

aS

ampl

e ty

peA

naly

ses

MS

Ref

eren

ces

Soi

l sam

ples

of t

ree

sub-

xeric

and

tree

mes

ic s

ites

in th

e no

rth a

nd in

the

sout

h bo

real

gro

win

g zo

ne

L, F

, H la

yer

NP

E, W

SE

, AS

, AIR

Tota

l C, N

, OM

, ash

IIR

yan

et a

l. 19

90

Soi

l sam

ples

of t

ree

sub-

xeric

and

tree

mes

ic s

ite

in th

e no

rth a

nd in

the

sout

h bo

real

gro

win

g zo

ne

L, F

, H la

yer

WE

C, W

EN

, wat

er-e

xtra

ctab

le

phen

ols

and

suga

rs

III(W

illia

ms

et a

l. 19

95).

Suo

min

en e

t al.

2003

, Yu

& D

ahlg

ren

2000

Woo

d &

Bha

t (19

88),

Dec

ompo

sitio

n ex

perim

ent

of fo

ur y

ears

in tr

ee s

ub-x

eric

and

tree

mes

ic s

ites

in th

e no

rth a

nd in

the

sout

h bo

real

gro

win

g zo

ne

Nee

dle,

bilb

erry

leav

es li

tter

mos

s

NP

E, W

SE

, AS

, AIR

Tota

l C, N

, OM

, ash

IR

yan

et a

l. 19

90

Litte

r fra

ctio

ns o

f L la

yer

of tr

ee s

ub-x

eric

and

tree

mes

ic s

ites

in th

e no

rth a

nd in

the

sout

h bo

real

gro

win

g zo

ne

Tree

, dw

arf s

hrub

and

mos

s lit

ter o

f L la

yer

NP

E, W

SE

, AS

, AIR

, OM

, ash

IR

yan

et a

l. 19

90

Juup

ajok

i and

Kiv

alo

soil

sam

ples

NP

E, W

SE

and

AS

frac

tions

of n

eedl

e, tr

ee a

nd m

oss

litte

r

of L

laye

r and

F a

nd H

laye

r sam

ples

Tota

l sug

ar a

nd s

ugar

com

pone

nts

of W

SE

and

AS

frac

tions

resi

n ac

ids,

fatty

aci

ds a

nd o

ther

extra

ctab

le li

pids

IVS

undb

erg

et a

l. (1

996)

and

Will

för e

t al.

(200

9).

Örs

å &

Hol

mbo

m (1

994)

.

Soi

l sam

ples

of s

ub-x

eric

and

mes

ic

site

of J

uupa

joki

AIR

frac

tion

of n

eedl

e, tr

ee a

nd

mos

s lit

ter o

f L la

yer

AIR

frac

tion

of F

and

H la

yer

Pyr

olys

is G

C/M

SV

Soi

l sam

ples

of s

ub-x

eric

and

mes

ic

site

of J

uupa

joki

Unf

ract

iona

ted

sam

ples

of n

eedl

e

tree,

and

mos

s lit

ter o

f L la

yer a

nd

F an

d H

laye

r sam

ples

Pyr

olys

is G

C/M

S

V

Soi

l sam

ples

of s

ub-x

eric

and

mes

ic

site

of J

uupa

joki

WS

E fr

actio

n of

L la

yer s

ampl

esP

yrol

ysis

GC

/MS

V

34

3.4.1 Detailed characterization of the water-soluble extractives (WSE), non-polar extractives (NPE), and acid-soluble (AS) fractions

In order to characterize the WSE fraction in detail, the water-extractable phenolic (III) concentration was determined by the Folin-Ciocalteu method (Suominen et al. 2003) and the concentration of soluble sugars by the method of Wood and Bhat (1988) (III). Water-extractable carbon (WEC) was analyzed using a carbon analyzer (Shimadzu TOC-5000A) and water-extractable N by fl ow injection analysis (FIA 5012) after oxidation of N to NO3-N with alkaline potassium persulphate (Williams et al. 1995). Total amount of sugar units in the WSE samples and the composition (IV) were analyzed by GC after acid methanolysis and derivatization according to Sundberg et al. (1996) and Willför et al. (2009) (IV). Identifi cation of individual components was performed by GC-MS analysis. The AS fraction was analyzed after neutralization by BaCO3. The total amount of monosaccharides was analyzed according to Sundberg et al. 1996. Triglycerides, diglycerides, and steryl esters in the NPE fraction were analyzed according to Örså and Holmbom (1994) (IV).

Pyrolysis with mass spectrometry is one of the best methods to study the biomacromolecules in soils and complex chemical composition of the soil organic matter (Saiz-Jimenez 1994, Dijkstra et al. 1998). To investigate if the WSE fraction contains lignin, the L layer samples were studied with pyrolysis GC-MS (V).

3.4.2 Detailed study of acid insoluble (AIR) fraction

Py-GC-MS analyses were performed on a fi lament pulse Pyrola 85 pyrolyzer (Pyrol AB, Lund, Sweden) connected to a GC-MS (HP 6890-5973 instrument). The unfractionated and AIR fraction of litter and F and H layer samples (V) were pyrolyzed at 650°C for 2 s. The identifi cation of the eluted compounds was done by comparing the spectra with the spectral library of Wiley 275, NIST 98 and spectral libraries of the Laboratory of Paper and Wood Chemistry, Åbo Academy.

3.5 Other chemical analyses

Litter samples collected from the L layer, the F layer, and the H layer were dried (60 °C) and weighed separately. The dry matter content was determined by drying sub-samples for 72 hours and litter material 24 hours in an oven at 105 °C (II). The organic (OM) matter content was determined as the loss in weight on ignition

35

in a muffl e furnace at 550 °C for 2 hours (I, II, III). The OM content was used for calculating the results per g OM. Total C and N using a CHN analyzer (Leco) (I, II).

3.6 Statistical analyses

The assumption of homogeneity of variances between the locations and site types was tested using Levene’s test, and the Kolmogorov-Smirnov test and normality plots were used to test normality of parameters used (II, III, IV). To assess whether the two locations (north and south boreal), and the two site types (mesic and sub-xeric) differed from each other in NPE, WSE, AS and AIR concentrations or amounts with respect to the accumulated litter stocks, the concentrations of organic fractions of the litter types and their stocks, the analyses of nested ANOVA was used (I). The two-way ANOVA was used to investigate the differences in NPE, WSE, AS and AIR concentrations between the north and south and the sub-xeric and mesic site types in different stages of decomposition (I). One-way ANOVA with Tukey’s test were used to investigate differences in NPE, WSE and AS fractions of samples (IV). To determine the differences in concentrations and amounts of C, N and carbon fractions and water-extractable compounds between site types, growing zones and soil layers, repeated measures ANOVA was used (II, III). All statistical analyses were conducted with SPSS 16.0 statistical software.

36

37

4 Results and discussion4.1 How does the chemical quality of litter differ between different

stages of decomposition in boreal coniferous soils?

The proportions of different organic fractions varied signifi cantly between the layers in the organic horizon (L, F, and H). AS (acid-soluble) fraction composed the largest fraction in the litter decomposition experiment (I), and in the L and F layers both by concentration and area basis, followed by AIR (acid-insoluble residue) (II). The carbohydrates in the AS fraction generally represent fragments of cellulosic plant material and are labeled as cellulosic carbohydrates (Rosenberg et al. 2003). In the H layer the AIR fraction was the most abundant (II). The concentrations of WSE and NPE fractions were highest in freshly fallen litter (I) and also signifi cantly higher in the L layer compared to the F and H layers (II). The fractionation method removed soluble polysaccharides in the WSE fraction and cellulose and hemicellulose-derived sugars in the AS fraction very effi ciently (III, IV, V), which supports earlier studies (Murata et al. 1998, Rosenberg et al. 2003).

4.1.1 Water-soluble compounds (WSE fraction)

In coniferous forest soils, the concentrations and stocks of water-extractable compounds generally decrease during the course of the decomposition process (Almendros et al. 2000, Karhu et al. 2010), because large amounts of soluble C are released from freshly fallen litter during the initial stages of decomposition (Don & Kalbitz 2005). The WSE fraction decreased quickly, and after two months in bilberry leaves and in one year in needle and moss litter reached the same level as found in dwarf shrub, tree and moss litter accumulated in the L layer (I). This is in a line with Sjöberg (2003), who found that organic fractions of decomposing needle litter reached the level of litter layer approximately in 1,5 years. Consistent with hypothesis, the concentrations of water-extractable compounds decreased along the decomposition continuum from the L to the F and from the F to the H layers (II, III, IV). The concentrations of total water-soluble C (III), sugars (III, IV), phenolics and WEN (III) decreased drastically from the L to the F layer, but only slightly from the F to the H layer (III).

Decreasing concentrations of phenols and sugars (III) from the L layer to the F and H layers indicate that water-soluble phenols from the L layer are not leached to deeper layers in the soil organic horizon. Earlier studies suggest that along with

38

the concentration, also the degradability of water-soluble C may decrease from the L to the H layer, as the degradation of recalcitrant substances often yields relatively stable and slowly-decomposable DOC (Don & Kalbitz 2005, Fröberg et al. 2006, 2007). The results are in line with earlier experiments which indicate that, in the early stages of decomposition, water-soluble or labile compounds in plant litter are more rapidly degraded than the insoluble AS and AIR fractions (Berg 2000, Girisha et al. 2003, Qualls & Haines 1992, Fröberg et al. 2003, Karhu et al. 2010). In addition to loss of labile compounds through decomposition (Lorenz et al. 2000, Karhu et al. 2010), these substances are also incorporated into microbial biomass (Balaria et al. 2009).

4.1.2 Extractable lipids (NPE fraction)

The concentration of the NPE fraction decreased during litter decomposition (I, II). Along with the concentration of NPE, also the chemical composition differed between the L, F and H layers. Detailed chemical characterization of the NPE fraction demonstrated that the concentrations of fatty acids (FAs) were lower in the F and H layers than in the L layer (IV). In addition, despite a wide variety of FAs detected in the L layer, only FAs of 12:0, 14:0, 16:0 and 18:0 were detected in the F and H layers (IV). A distribution of long-chain FAs, ranging from C16 to C32 is commonly observed in acid soils covered by higher plant vegetation (e.g. Amblés et al. 1994, Almendros et al. 1996, van Bergen et al. 1997, Rosenberg et al. 2003), and short chain FAs are more microbial than plant origin (Amblés et al. 1994, Almendros et al. 1996). The FAs found in the H layer (IV) indicate microbial origin (Grasset & Amblés 1998, Buurman et al. 2008) and are relatively slowly decomposable (Fjalal & Read 1983, Saiz-Jimenez 2005) and thus may accumulate in acidic soils (Nierop et al. 2005). The composition of FAs found in this study were in line with earlier studies on lipid extracts in coniferous materials (Fernández de Simón et al. 2001, Rosenberg et al. 2003).

The resin acid compositions in the litter samples (IV) were in line with previous studies in coniferous forests with abundant ericaceous dwarf shrubs, although some compounds identifi ed in the fresh litter, such as neoabietic acid and pimaric acid (Dijkstra et al. 1998, Fernández de Simón et al. 2001, Kainulainen & Holopainen 2002, Kanerva et al. 2008) were absent. The concentration of resin acid appeared to decrease from the L layer to the F and H layers, but the difference was not statistically signifi cant (IV). However, the composition of resin acids changed from the L to the F and H layers. The proportion of dehydroabietic acid and its

39

oxidized forms relative to other resin acids was higher in the F and H layers, which demonstrates their strong tendency to accumulate in boreal forest soils (IV, V). These results agree well with a decomposition experiment conducted by Kainulainen and Holopainen (2002) showing very rapid decomposition rates of other resin acids and accumulation of dehydroabietic acid along with the litter decomposition. Some studies suggest that resin acids do not contribute to the chemical composition of SOM to a large extent (Dijkstra et al. 1998, Kanerva et al. 2008), whereas others suggest that especially dehydroabietic acid is rather stable and accumulates in soils and sediments (Leppänen & Oikari 2001). Dehydroabietic acid may be recalcitrant to microbial degradation due to its aromatic structure and may refl ect its function in trees as a protective substance against wood-rotting fungi (Henriks et al. 1979, Harju et al. 2002).

Total concentration of sterols in the NPE fraction remained unchanged from the L to the F and H layers, but as the concentration of other substances decreased, sterols and oxidized forms of these were much more abundant in the F and H layers than in the L layer (IV, Fig 6). Steroids and their degradation products have frequently been identifi ed in soil and leaf/litter extracts (van Bergen et al. 1997, Bull et al. 1998, 2000, Kanerva et al. 2008, Valentín et al. 2010). Kanerva et al. (2008) have shown that in coniferous soils triterpenes like beta-sitosterol and sitosterol are common in, e.g. coniferous needles, Vaccinium species, and in the L, F, and H layers. Steroids are also commonly synthesized by soil fungi (Grandmougin-Ferjani et al. 1999). The parallel decrease in sterol content from the L to H layer and an increase of 7- oxositosterol and sitostadiene (IV) indicates their degradation by enzymes (Valls et al. 2009). Naafs et al. (2004) found drastically decreasing steroid concentrations along the soil profi le in andisols, but Dijkstra et al. (1998) and Kanerva et al. (2008) found increasing concentrations of sterols in relation to resin acids along the soil profi le in boreal coniferous forests soils.

4.1.3 Cellulose and hemicellulose (AS fraction)

The concentration of AS increased from the L to the F layer, and decreased from the F to the H layer (II). The same trend was found in the decomposition trial of bilberry leaf, needle and moss litter (I). Pyrolysis-GC analyses were in line with the fractionation results, showing high abundance of anhydrosugar and acetic acids, which are typical pyrolysis products of cellulose (V). The AS fraction is generally an abundant fraction in litter and soil material, its abundance in relation to AIR depending on the soil type or litter decomposition stage (Hobbie 1996, Weintraub

40

& Schimel 2003, Tian et al. 2000). Put together, my results confi rm that degradation products of cellulose accumulate in F and H layers (IV, V, II). However, I found a peak in the AS concentrations in the F layer (II), which supports the assumption that long-chain carbohydrates are more slowly decomposable than compounds in extractable (NPE and WSE) fractions, but decompose at a faster rate than the AIR fraction (Berg 2000, Hobbie 1996). A major part of the cellulose in plant material is protected by lignin, and accessible for microbial degradation only after lignin degradation (Jennings & Lysek, 1996, Niemenmaa 2008). During the second phase of litter decomposition, both cellulolytic and lignolytic enzymes are active (Berg 1991, Kalbitz et al. 2006, Niemenmaa 2008). Lignin degradation by white-rot fungi is believed to be a co-metabolic process also requiring other carbon sources than lignin, because the microorganisms generally do not use macromolecular lignin as a sole carbon source (Kirk & Farrell 1987, Hatakka 2001). This extra carbon is generally derived from cellulose and hemicelluloses.

Detailed characterization of the AS fraction showed that the concentration of glucose remained stable while concentration of other sugars decreased from the L to the H layer (IV, Fig 2). Glucose was the most common sugar found in the AS fraction (IV, Fig. 2, I, II) at each stage of decomposition, which indicates that along the decomposition continuum, the importance of cellulose in relation to hemicelluloses as a source of glucose increases (Curling et al. 2001, Berg & McClaugherty, 2003).

4.1.4 Acid insoluble residue (AIR)

Boreal forest trees and ericaceous understory plants produce litter rich in lignin and phenolic secondary metabolites (Gallet & Lebreton 1995, Hobbie 1996, Wardle et al. 2003, Kraus et al. 2004, Vargas et al. 2006, Kanerva et al. 2008). The AIR fraction increased along with litter decomposition (I) and from the L to the H layer (II). These results were in line with earlier decomposition trials (Almendros et al. 2000, Tian et al. 2000, Loya et al. 2004) and decomposition experiments conducted with SOM (Shaver et al. 2006, Weintraub & Schimel 2003, Karhu et al. 2010). However, increasing AIR concentrations do not necessarily refl ect accumulation of lignin. Boberg (2009) showed an increase in the AIR fraction at the same time when no apparent lignin loss from the AIR fraction was detected. The reason for an increase in the AIR is unknown and may refl ect either accumulation of lignin-like or other recalcitrant compounds. Niemenmaa (2008) and Martínez et al. (2010)

41

found that brown-rot fungi predominantly utilize hemicellulose and cellulose leaving behind a chemically modifi ed lignin residue.

More detailed characterization of the AIR fraction (V) showed that in the litter samples, the AIR fraction contained some compounds derived from carbohydrates, such as furfural (Marinari et al. 2007, Buurman et al. 2005, 2007) and other furans, which are linked with cellulose degradation (Alén et al. 1996, Nierop et al. 2001). Degradation-resistant carbohydrates are mostly believed to be of microbial origin (Dijkstra et al. 1998, Buurman et al. 2005, Li et al. 2004), but could also originate from plant debris. Thus, carbohydrates found in the AIR fraction of the F and H layer (V) most probably indicate carbohydrates modifi ed by microbes during decomposition (Almendros et al. 2000).

Generally phenols are common pyrolysis products of SOM, and can be derived from several types of biopolymers such as protein, polycarboxylic acids, carbohydrates or lignin (Dijkstra et al. 1998, Nierop et al. 2001, Buurman et al. 2005). The relatively high content of guaiacols in the AIR fraction suggests that the phenolic structures of the soil samples were most likely derived from angiosperm lignin (Crawford & Olson 1978, Saiz-Jimenez & De Leeuw 1986, Preston et al. 1994). Also some other phenols, such as catechol and methoxycatechol, have been reported to form during degradation of guaiacyl lignin (Lu et al. 2000, van Bergen et al. 2000). For example, lignin in pine trees is almost totally composed of coniferyl alcohol units, which produce mostly guaiacols in pyrolysis (Nierop et al. 2001). An increase of lignin monomers such as 4-vinylguaiacol, 4-methylguaiacol and guaiacol from litter material to F and H layer samples (V) and vanillin and vanillic acid and oxidized forms of lignin monomers indicate that lignin is at a more degraded stage in the F and the H layers than in the forest litter (Preston et al. 1994). Li et al. (2006) have shown that pyrolyis of humic acids especially yielded guaiacyl derivatives, coniferyldehyde and coniferyl alcohol, which were detected more in H layer samples (V) with the highest degree of humifi cation. Due to the variable composition of AIR along the decomposition gradient, my results do not support the idea that lignin accumulates along with the process of decomposition (Berg 2000). However, lignin was found at a degraded stage (Rosenberg et al. 2003, Sjöberg 2003, Bahri et al. 2008, Lima et al. 2008, Boberg 2009, Martínez et al. 2010), and degradation products of lignin and other polyphenols constitute a major proportion of the accumulated SOM (Kalbiz et al. 2006, Bahri et al. 2008, Martínez et al. 2010).

The relative abundance of lipids in the AIR fraction was higher in the H layer than in the litter samples, as in the H layer, and some FAs and resin acids were also

42

detected (V). The relative decrease of polyphenol-derived compounds in relation to other compounds was most signifi cant in the H layer sample of the mesic site. A decrease in polyphenol-derived compounds may suggest that spruce lignin is more labile than pine lignin (Kalbitz et al. 2006). However, I detected high abundance of methyldehydroabietate, which is a degradation product of microbial oxidation of dehydroabietic and isopimaric acids (Vorob’ev et al. 2001). Put together, my studies suggest that dehydroabietic acid (IV) and its oxidized form methyldehydroabietate (V) may have high importance in the accumulated SOM. In the F and H layers, some low molecular weight FAs were also detected in the AIR fraction (V), which are often found in the humic substance pyrolysis or hydrolysis products (van Bergen et al., 1997, Grasset & Amble`s, 1998, Naafs & van Bergen 2002, Buurman et al. 2008). The fact that they were detected only in the AIR fraction of F and H layers suggests that they may be produced by brown-rot and white-rot fungi during the breakdown of lignin and cellulose (Dijkstra et al. 1998, Hatakka 2001).

Some earlier studies have indicated that compounds, especially hydroxy- and epoxy fatty acids derived from cutin and suberin from leaves and roots may accumulate in the AIR fraction (Preston et al. 1997) and soils (Bernards 2002). Otto and Simpson (2006) found that hydroxy- and epoxy fatty acids from the cutin of leaves are relatively easily decomposed. I did not fi nd any compounds originating from these plant constituents, but they could have been detected using other methods.

4.1.5 A synthesis: How does the litter quality infl uence the SOM quality, and is recalcitrant carbon derived from lignin, other polyphenols, lipophilics, or carbohydrates?

High concentrations of anhydrosugar and furans found in unfractionated litter, F and H layer samples (V), and high concentration of the AS fraction in all layers (I, II) are in line with previous investigations showing the importance of substances derived from polysaccharides and other carbohydrates in accumulating soil OM (de Leeuw & Largeau 1993, Almendros et al. 2000, de Leeuw et al. 2006, Nierop & Buurman 2001, Kögel-Knabner 2002, Buurman et al. 2005). In line with earlier studies (e.g. Rumpel et al. 2005, Otto & Simpson 2006, Buurman et al. 2005), compounds derived from lignin or other polyphenolics composed a signifi cant proportion of the unfractionated and AIR fraction of litter and soil samples (V). Although the concentration of AIR increased with litter decomposition (I) and from the L layer to the H layer (II), detailed analyses showed that the composition of the

43

AIR differs between the litter and the F and H layers. In the L layer, the AIR fraction was composed of phenolics and some carbohydrates, but in the F and H layers, the AIR fraction was a mixture of compounds derived from phenolics and lipophilic compounds. My analyses do not enable assessing the role of root litter production, as in the H layer a proportion of the organic compounds originate from root litter (Nierop et al. 2001, Otto & Simpson 2006, 2007) and not from aboveground plant litter. The concentrations of NPE and WSE fractions decreased in relation to decomposition (I, II). However, my results show that in addition to concentrations, also the composition of the soluble fractions changes with decomposition. In the F and H layers, the NPE fraction is composed of a much shorter range of compounds than in the L layer (IV). Thus, my results demonstrate that although the proportion of the soluble fractions decreases, some specifi c lipophilic compounds, such as dehydroabietic acid, are important components of accumulating SOM.

4.2 How does the climate affect decomposition rate of organic fractions?

The quality and quantity of soil C accumulation could be explained by both the litter input and the litter decomposition rates. I therefore tested whether the quality of fresh litter and differences in litter decomposition rates between the northern and the southern research areas are refl ected in the quantity and quality of the litter accumulated on the forest fl oor (I). Earlier studies have shown that the concentration of phenolics in several boreal understory dwarf shrubs and trees is higher in the north than in southern boreal forests (Hokkanen et al. 2009, Martz et al. 2009, 2010, Stark et al. 2008, Åkerström et al. (2010). I therefore predicted that the fresh litter input would be more recalcitrant to decomposition in the north than in the southern boreal forests. In contradiction with predictions, I found no difference in the initial concentrations of organic fractions by location (I). However, the concentrations of phenolics in the accumulated litter was higher in the north than south (III). These results suggest that even in cases where there is no difference in the total concentration of the WSE fraction, there still may be changes in the compound composition within each fraction.

I also predicted that during the course of litter decomposition, soluble substrates would decompose more rapidly in the south than in the north, leaving higher concentrations of recalcitrant organic fractions in the litter in the south (Berg et al. 1993, Coûteaux et al. 1995, McTiernan et al. 2003). However, loss of dry weight and C loss during a 4-year litter decomposition experiment did not differ between

44

the north and the south and after four years decomposition there was no difference in organic fraction concentrations between north and south (I). The overall decomposition rate of needle litter tended to be lower in the north than south, and the WSE fraction of bilberry leaves decomposed at a lower rate in the north than in the south at the beginning of decomposition (I). Although climate has been regarded to control the decomposition rates at high latitudes (Coûteaux et al. 1995, Johansson et al. 1995), these results suggest that the temperature difference between northern and the southern boreal forests does not infl uence the overall decomposition rate or the relative decomposition rates of individual organic fractions (I). Furthermore, my results are in contrast to some other studies suggesting that the temperature-dependence of decomposition varies among the different compound groups (e.g. Osono & Takeda 2006). Rather, the results of this study support the view that soil microbial respiration may be relatively indifferent to changes in temperature, because the soil microorganisms in northern environments may be adapted to low soil temperature (Haimi et al. 2005, Bradford et al. 2008). Prescott (2005) argued that the correlation with decomposition is likely to increase with the increasing range of the variable, and thus the factor with greatest range is likely to become the best predictor of the decomposition rate. The role of climate may therefore be obvious along large gradients, where both the climate and the vegetation zone change, but not within the boreal zone with a 4–5 °C temperature difference during the last 30 years in addition to relatively similar humidity levels. Other factors than temperature may also affect the decomposition rate like seasonality of rainfall, differences in solar radiation or soil nutrient status (Pausas 1997).

Although I detected no difference in the decomposition of organic fractions in the litter decomposition experiment (I), in the decomposition continuum, the concentration of AIR was lower in the north than south boreal forests in the F and H layer, although there was no difference in the L layer (II). This indicates that AIR accumulates in relation to other organic fractions to a greater extent in the south than in the north, which may indicate differences in microbial activity during the decomposition process (Anderson & Hetherington 1999, Li 2010). It should be borne in mind that the results of proximate analyses are relative, and higher WSE concentration is related to lower AIR concentration in the north boreal site and higher AIR and lower WSE in the south (II). The changes in the organic matter fractions usually coincide with changes in the microbial community composition, and especially the proportion of labile and recalcitrant compounds seems to have an important role (Pennanen et al. 1999, Osono & Takeda 2006, Allison et al. 2009, Karhu et al. 2010, Song et al. 2010). The different outcome of the litter

45

decomposition experiment (I) and the comparison of L, F, and H layers suggest that initial litter decomposition cannot explain the carbon dynamics in soils, as already suggested by, e.g. Prescott et al. (2004a, 2005b).

4.3 How does soil fertility affect decomposition of organic fractions?

Berg and Staaf (1981) suggested that N may limit microbial growth especially at the early stages of decomposition. Because of that the decomposition of litter is more rapid in nutrient-rich than in nutrient-poor sites. Allison et al. (2009) suggest that the addition of N positively affects decomposition of substrates with low N content like cellulose and wood substrates. However, some studies suggest that high N concentration may decrease the decomposition of lignin or other recalcitrant substances (Fog 1988, Berg & McClaugherty 2003, Sjöberg 2003). In this study, the N concentrations within all litter types and soil layers were higher in the mesic than in the sub-xeric sites, as well as in the southern than in the northern sites (I, II). Also the freshly fallen litter and soil organic matter of L, F and H layers in sub-xeric sites had higher C/N and AIR/N than in mesic sites (I, II ), except for the AIR/N ratio in freshly fallen moss litter, which was higher in mesic than sub-xeric sites at the north boreal growing zone (I). However, this seemed to have no consequences on the overall decomposition rates within a litter type. Bilberry leaves with the highest concentrations of N and WSE showed the fastest decomposition rate, and the moss litter with the lowest concentration of N and WSE showed the slowest decomposition rate (I), which is in line with the argument that a higher initial N or WSE concentration and lower lignin or AIR concentration should produce a faster decomposition rate, based on comparisons among different litter types (Berg & Tamm 1991, Prescott, 1995, 2000, Tian et al. 2000, Wardle et al. 2003).

The results of the litter decomposition trial (I) confi rmed earlier fi ndings that, in similar conditions (soil type, climate), different litter types decompose at greatly varying rates (Wardle et al. 2003, Fioretto et al. 2005, Hättenschwiler & Gasser 2005, Cornwell et al. 2008). The decomposition rate of bilberry leaves with high WSE concentrations was extremely rapid and of the same order of magnitude reported in previous experiments (e.g. Hobbie, 1996, Johansson 1993). By contrast, litter with low concentrations of carbohydrates or high concentrations of lignin generally decompose at a lower rate (Hobbie 1996, Prescott et al. 2004, Fioretto et al. 2005, Hobbie et al. 2006). Even though the differences in initial litter quality do not affect decomposition rate of different litter types between sub-xeric and mesic

46

sites or between north and south (I), the quality of plant litter was refl ected in the quality of the SOM at a late stage of decomposition (II). The present study indicates that short-term incubation studies or decomposition rate do not always refl ect the long-term decomposition process (Prescott 2000, Prescott et al. 2004, 2005).

4.4 Determinants of soil carbon stocks in boreal forest soils

The composition and total amount of C stocks in the soil are determined by both the quantity and quality of the litterfall and the conditions under which litter and soil OM decomposition take place (Kalbitz et al. 2006, Sariyildiz et al. 2005). Total C stocks were signifi cantly higher in the mesic than in sub-xeric forests, and higher in the south than in north boreal forests (II). Litterfall from trees is higher in south than north boreal forests (Berg et al. 1995, Vucetich et al. 2000, Ukonmaanaho et al. 2008), However, I found substantial differences in the composition of the accumulated litter stocks: In the south boreal forests, the tree litter constituted the most important C stock in the L layer, but in the north boreal forests, the amount of moss litter equaled that of the tree litter in the sub-xeric sites (I), and outweighed that of the tree C stock in the mesic sites (I). Consequently, the total litterfall does not decrease toward the north to the same extent than the litterfall from trees, because of increasing quantities of litter from the understory vegetation (Persson 1980, Preston 2006). My fi ndings suggest that the differences in the quality of litter and amount of soil OM between the south and north boreal forests (II) results from differences in the plant species composition (Almendros et al. 2000, Quideau et al. 2001, Merilä et al. 2010), and in quantitative differences in the litter input by various growth forms (Davidson & Janssens 2006), rather than from differences in the litter quality within litter type or decomposition rates (I).

Based on earlier studies Norway spruce forests, in general, annually accumulate more C in soils than the pine forest mainly corresponding to the amount of litterfall in these forests (Akselsson 2005, Stendahl et al. 2010), although quantitative differences in the litter production between the different forest types are not always directly refl ected in the total organic C stocks (Preston et al. 2006). The litter decomposition trials (I) demonstrated that V. myrtillus leaves decomposed at a rapid rate and therefore are unlikely to contribute to the soil C stocks to a considerable extent. Moreover, at some study sites herbs and grasses were abundant in the ground vegetation (II), but the amount of their litter was negligible in the accumulated litter in the L layer (I). Therefore, some plant growth forms do not seem to contribute to the accumulated SOM (Kang et al. 2009). Mosses, by contrast, decomposed at a

47

very slow rate, and for this reason, contribute considerably to OM accumulation in boreal forests (Hobbie et al. 2000). The differences in the quantity of the different litter types suggest that the trees, the understory dwarf shrubs, or the mosses, contribute to soil C accumulation to a varying extent in different boreal climatic zones.

The total C storage in the sub-xeric forests varied greatly between the climatic zones, but in the mesic forests there were no differences in the C storage between the north and the south (II). Correlation analysis between the parameters of the L layer and the stocks of C in the F and H layers showed a positive correlation between the total C stocks and N concentrations on the mesic sites, and a positive correlation between the C stocks and concentrations on the sub-xeric sites (II). This indicates that the factors regulating OM accumulation may vary according to the nutrient level of the forest site type, although regression analyses have to be interpreted with caution as they do not indicate cause and effect (Prescott 2005).

When water-extractable compounds were expressed as stocks in the SOM, the total stock of water-extractable C and N increased from L to F and from F to H layers (III). Some previous studies have suggested that DOC is mainly derived from F and H layers (Solinger et al. 2001, Fröberg et al. 2007). Neff et al. (2000) found that DOC and DON fl uxes are closely related to stocks of soil organic matter. The DOC concentrations in soil waters in the same study sites during 2001–2004 did not differ between the south and the north, or between the site types (Derome et al. 2007). When calculated on an areal basis, the amount of leached DOC did not differ between the north and the south on the mesic sites, but the amount of leached DOC was considerably lower in the north than in the south on the sub-xeric sites (Lindroos et al. 2008). The amount of leached DOC was in proportion to the total C stocks because the total C stocks on the mesic plots did not differ between the north and the south, but were signifi cantly lower in the north on the sub-xeric sites (II, III). These results are in line with the previous fi ndings of Fröberg et al. (2006) and Liechty et al. (1995), who reported that the amount of DOC leaching from the organic horizon decreased with latitude and was in proportion to the total soil C stocks (Fröberg et al. 2006). In agreement with the results of earlier investigations (Smolander and Kitunen 2002, Kiikkilä et al. 2006) the total stocks of WEC and WEN were signifi cantly higher on the mesic than in the sub-xeric plots refl ecting the size of total C stock in the organic layer (II).

48

49

5 Conclusions and future perspectivesThe results of this study suggest that the differences in plant community structure and quantitative differences in the litter input by various growth forms, especially the relative abundance of plant growth forms that produce recalcitrant litter (trees, mosses) between the north and south boreal forests and the mesic and sub-xeric sites, has a great impact on OM accumulation, whereas differences in the litter quality or decomposition rates within each litter type have only a minor infl uence. The impact of initial N content of litter on SOM accumulation remains open. However, litter with a high initial WSE and N concentration decomposed at a faster rate than litter with a lower initial N and WSE concentration at the fi rst stage of decomposition, irrespective of the soil fertility or climate conditions.

In sub-xeric sites, the soil C stocks were considerably higher in the south than north boreal forests, but there was no difference between the north and the south in the mesic sites, which supports the conjecture that on a regional scale litter production is a more important factor determining SOM accumulation than climatic factors. By contrast, concentrations of organic fractions, especially the concentrations of labile carbon compounds, seem to be poor indicators of soil C stocks, if the quantitative proportions of each fraction on an areal basis are not taken into account. The acid-insoluble residue (AIR), which contains the most recalcitrant compounds, increased with the litter decomposition. However, the composition of AIR also changes from the litter materials to the F and H layers in the soil organic horizon. In the L layer, when litter is at an early stage of decomposition, the concentration of AIR refl ects concentrations of lignin and other insoluble polyphenols in the litter. In the F and H layers, lignin and other polyphenols can be found at a more degraded state, but lignin-derived and chemically modifi ed polyphenolics constitute a large proportion of the AIR. However, the amount of lipophilic compounds, which mainly results from accumulation of resin acid methydehydroabietate, in the AIR increases. Earlier it has been shown that the AIR also contains a major aliphatic component (Zech et al. 1997, Preston et al. 1997), which can be detected with solid-state 13C NMR spectroscopic analysis. On the other hand, although the different litter types decomposed at different rates and showed very different AIR concentrations, they showed relatively similar composition of the AIR. Furthermore, despite differences in the ground vegetation and main tree species (Norway spruce, Scots pine), the pyrolysis spectra of the F and H layers showed only minor differences between a mesic and a sub-xeric site. This supports the suggestion by Melillo et al. (1989) that plant litter material becomes more similar during the decomposition process despite litter origin.

50

The fractionation method by Ryan et al. (1990) is able to separate extractable compounds and cellulose from the AIR fraction well. If detailed information on the composition of the AIR fraction is needed, pyrolysis-gas chromatography/mass spectrometry is a useful tool. For separation of lignin from other polyphenols, other methods, such as CuO oxidation (Otto & Simpson 2006, 2007) are needed. Comparison of the phenols in the AIR fraction and CuO extracts with lignin-rich (tree, needle) and lignin-poor (moss) SOM may provide a tool to distinguish lignin-derived phenols from other polyphenolics, which are generally found in a modifi ed stage in organic materials. The soluble extract contains free aliphatic lipids mainly derived from higher plants, but if information of ester–bound lipids like suberin is needed, it would be useful to conduct also base hydrolysis (Otto & Simpson 2007). Pyrolysis and CuO oxidation methods would also allow to detect the aliphatic degradation products, if they are considered in the analyses (Dai 2002, Rosenberg et al. 2003, Otto & Simposon 2007, Buurman et al. 2008).

The ability of the microbial community to use different carbon fractions as an energy source would be an important subject for future investigations in both laboratory and fi eld conditions. Furthermore, because F and H layer material in boreal forests also contains decomposition results of plant roots and rhizomes, the contribution of roots and rhizomes to SOM accumulation would also require further investigation.

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Original articlesI Hilli S, Stark S & Derome J (2010) Litter decomposition rates in relation

to litter stocks in boreal coniferous forests along climatic and soil fertility gradients. Applied Soil Ecology 46: 200–208.

II Hilli S, Stark S & Derome J (2008) Carbon quality and stocks in organic horizons in boreal forest soils. Ecosystems 11: 270–282.

III Hilli S, Stark S & Derome J (2008) Qualitative and quantitative changes in water-extractable organic compounds in the organic horizon of boreal coniferous forests. Boreal Environment Research 13(supp.B): 107–119.

IV Stark S, Hilli S, Willför S, Smeds A, Reunanen M, Penttinen M & Hautajärvi R (2010) Composition of lipophilic compounds and carbohydrates in the accumulated plant litter and soil organic matter in boreal forests. Submitted manuscript.

V Hilli S, Stark S, Willför S, Smeds A, Reunanen M & Hautajärvi R (2010) What is the composition of AIR? Chemical characterization of litter and soil in two south boreal forests using sequential fractionation and pyrolysis- GC-MS. Submitted manuscript.

Reprinted with kind permission for the publishers Elsevier (I), Springer Science and Business Media (II) and Boreal Environment Research Publishing Board (III). Original articles are not included in the electronic version of the dissertation.

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