Maria J.I. Briones

DECOMPOSITION OF EUCALYPTUS LEAVES IN LITTER MIXTURES

Abstract Leaf litter from Eucalyptus globulus was decomposed alone and in mixture with either oak (Quercus petraea), ash (Fraxinus excelsior) or birch (Betula pendula) leaf litter under laboratory conditions. Decomposition was monitored as CO2 release and leaching of inorganic N over 13 weeks. At the end of the experiment, litters were separated into their species components and analyzed for mineral composition (K, Ca, Mg, P and N) and mass loss. Differences between expected and measured rates of decomposition were evaluated, based on a comparison between the results from the pure litters and the mixtures. Mixing eucalyptus litter with oak litter resulted in enhanced total CO2 release from the litter mixture when compared with the pure components. Similar, but less marked, positive interactions were observed in mixtures with eucalyptus + birch and eucalyptus + ash. Decomposition of eucalyptus litter in the presence of the other litters also influenced N mineralisation, resulting in greater net N retention in the mixtures with eucalyptus + oak and eucalyptus + birch, but a decrease in mixture with ash. The results support the conclusion that the decomposition of litters in mixtures cannot be readily predicted from the behaviour of the component litters decomposing in isolation. We suggest that mixtures of eucalyptus with other litters could be one mechanism by which the high productivity rates of eucalyptus plantations may be maintained, and that manipulation of litter mixtures could assist in synchronising nutrient release and plant uptake.

Effects of climate change on soil fauna; responses of enchytraeids, Diptera larvae and tardigrades in a transplant experiment

Abstract A transplant experiment was carried out on the Moor House National Nature Reserve, Cumbria, UK, and two sites with different climatic characteristics were selected for determining how Enchytraeids, Diptera larvae and Tardigrades respond to changes in climate. The soil was a cambic stagnohumic gley, characterized by a high organic matter content and low pH. Vegetated soil cores were taken from near the summit of Great Dun Fell (GDF) and used for the transplant experiment. Cores were then placed back at GDF (845 m) and also transplanted to Sink Beck (SNK, 480 m), providing a mean annual temperature increase of 2.5°C. In order to discriminate between the effects of temperature and rainfall on the size and vertical distribution patterns of the fauna, a further series of lysimeters were established at SNK, and rainfall inputs were manipulated, intending to provide a total input equivalent to the ambient rainfall at the top site. At regular intervals, a number of cores were destructively sampled from each treatment (top site—GDF; lower site—SNK; enhanced rainfall inputs-SNK) and the vertical distribution of the soil fauna determined. Enchytraeidae, Tardigrada and Diptera were commonly found in soil samples at the Reserve and appeared sensitive to the imposed changes in temperature and moisture. Different species of enchytraeids responded differently: numbers of Cognettia sphagnetorum were correlated positively with temperature, whereas their vertical distribution was determined by moisture. Cernosvitoviella atrata was unable to avoid dry conditions which apparently caused severe mortality when exposed to the elevated temperatures; Achaeta eiseni was a more tolerant species, increasing in numbers with increasing temperature. Diptera larvae appeared to be dependent on the moisture status of the upper soil layers and their populations were reduced at higher temperatures and lower moisture contents. By contrast, tardigrades were able to survive adverse conditions by entering an anhydrobiotic stage, recovering when seasonal climate improved. General trends in soil faunal responses to climatological changes in the UK are predicted.

ELEVATED CO2 AFFECTS FIELD DECOMPOSITION RATE AND PALATABILITY OF TREE LEAF LITTER: IMPORTANCE OF CHANGES IN SUBSTRATE QUALITY

could be related to changes in tissue quality resulting from growing the plants at higher CO2 concentrations, with C-to-N ratios and lignin contents being significantly increased. The elevated CO2 treatment also aAected the rate of consumption of ash leaf litter by Oniscus asellus L. (Isopoda: Oniscoidea), with significantly less (ˇ16%) material being consumed for litter derived from the high CO2 regime. Our results indicate that changes in litter quality, which we may expect under elevated CO2, may aAect litter palatability for soil fauna. # 1998 Elsevier Science Ltd. All rights reserved

Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands

Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.

Vegetation exerts a greater control on litter decomposition than climate warming in peatlands

Historically, slow decomposition rates have resulted in the accumulation of large amounts of carbon in northern peatlands. Both climate warming and vegetation change can alter rates of decomposition, and hence affect rates of atmospheric CO2 exchange, with consequences for climate change feedbacks. Although warming and vegetation change are happening concurrently, little is known about their relative and interactive effects on decomposition processes. To test the effects of warming and vegetation change on decomposition rates, we placed litter of three dominant species (Calluna vulgaris, Eriophorum vaginatum, Hypnum jutlandicum) into a peatland field experiment that combined warming.with plant functional group removals, and measured mass loss over two years. To identify potential mechanisms behind effects, we also measured nutrient cycling and soil biota. We found that plant functional group removals exerted a stronger control over short-term litter decomposition than did approximately 1 degrees C warming, and that the plant removal effect depended on litter species identity. Specifically, rates of litter decomposition were faster when shrubs were removed from the plant community, and these effects were strongest for graminoid and bryophyte litter. Plant functional group removals also had strong effects on soil biota and nutrient cycling associated with decomposition, whereby shrub removal had cascading effects on soil fungal community composition, increased enchytraeid abundance, and increased rates of N mineralization. Our findings demonstrate that, in addition to litter quality, changes in vegetation composition play a significant role in regulating short-term litter decomposition and belowground communities in peatland, and that these impacts can be greater than moderate warming effects. Our findings, albeit from a relatively short-term study, highlight the need to consider both vegetation change and its impacts below ground alongside climatic effects when predicting future decomposition rates and carbon storage in peatlands.

Enhancing the understanding of earthworm feeding behaviour via the use of fatty acid δ13C values determined by gas chromatography-combustion-isotope ratio mass spectrometry†

Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.

Stable-isotope labeling and probing of recent photosynthates into respired CO2, soil microbes and soil mesofauna using a xylem and phloem stem-injection technique on Sitka spruce (Picea sitchensis).

RATIONALE Here we report on the successful application of a novel stem-injection stable-isotope-labeling and probing technique in mature trees to trace the spatial and temporal distribution of rhizosphere carbon belowground. METHODS Three 22-year-old Sitka spruce trees were injected with 6.66 g of (13)C-labeled aspartic acid. Over the succeeding 30 days, soil CO(2) efflux, phospholipid fatty-acid (PLFA) microbial biomarkers and soil invertebrates (mites, collembolans and enchytraeids) were analyzed along a 50 m transect from each tree to determine the temporal and spatial patterns in the translocation of recently fixed photosynthates belowground. RESULTS Soil δ(13)CO(2) values peaked 13-23 days after injection, up to 5 m from the base of the injected tree and was, on average, 3.5‰ enriched in (13)C relative to the baseline. Fungal PLFA biomarkers peaked 2-4 days after stem-injection, up to 20 m from the base of the injected tree and were (13)C-enriched by up to 50‰. Significant (13)C enrichment in mites and enchytraeids occurred 4-6 days after injection (by, on average, 1.5‰). CONCLUSIONS Stem injection of large trees with (13)C-enriched compounds is a successful tool to trace C-translocation belowground. In particular, the significant (13)C enrichment of CO(2) and enchytraeids near the base of the tree and the significant (13)C enrichment of PLFAs up to 20 m away indicate that mature Sitka spruce (Picea sitchensis) have the capacity to support soil communities over large distances.

Natural abundance of 13C and 15N in earthworms from different cropping treatments

Summary In order to get a better understanding of soil fauna responses to land use changes we used an experimental design established in Devon (UK) and selected five replicated treatments: maize (C4 plant), grass (C3 plant), clover (C3 plant fixing N from the air) and two bicropping systems consisting of maize with a clover understorey. Soil and earthworm samples were taken from each treatment in two sampling occasions (July and October 1998). Results showed that in all treatments the highest C and N contents were measured in the top 10 cm of the soil, with the exception of the maize plots possibly as result of recent rotavation. In contrast soil delta values increased with depth suggesting that agricultural practices affect the distribution of the organic matter through the soil profile. Isotopic composition of the earthworms showed that they were also significantly affected by the cropping systems. In agreement with previous findings N isotope values seem to be related to ecological groupings and ontogenic changes. Furthermore, under bicropping systems endogeic worms seem to gain their nutrition from older residues which is also in correspondence with their vertical distribution in the soil.

A Dynamic Study of Earthworm Feeding Ecology Using Stable Isotopes

Changes in the specific diet of earthworms with time in relation to landuse changes and two different climates were studied by analysing (13)C and (15)N natural abundance in soils and animals. Soil samples from three depths (0-10, 10-20 and 20-30 cm) and earthworms were collected from two sites: Santiago (Northwest Spain) and North Wyke (Southwest England) both consisting of replicated long-term grasslands and recently converted to maize plots. Earthworms were hand-sorted in the field at the peak of the maize growth and after harvesting at both sites. In the Spanish plots, nine and eight earthworm species, all belonging to the three ecological categories (epigeic, anecic and endogeic), were found under maize and permanent pasture, whereas at the English site five and seven different species were, respectively, identified. At both sites (13)C isotopic values of the earthworm tissues reflected changes in diet from C(3) to C(4) with epigeic and epi/anecic worms in the maize plots showing one delta unit difference in relation to the ones found in the grassland plots. Anecic worms seemed to be less responsive to landuse changes. The higher (13)C values of the Spanish soils were also reflected in the earthworm tissues when compared with the English samples. (15)N values showed no clear relationship with the cropping treatments but were clearly related to the ecological grouping, with endogeic worms reaching the highest values whereas for the epigeic and epi/anecic species the lowest values were obtained. This finding was also previously recorded by other authors1 and suggests that, in the future, stable isotope techniques could also be a useful tool in taxonomic studies. Copyright 1999 John Wiley & Sons, Ltd.

Is tagging with visual implant elastomer a reliable technique for marking earthworms

Visual implant elastomer (VIE) has recently been employed to investigate different aspects of earthworm ecology. However, a number of fundamental questions relating to the detection and positioning of the tag, its persistence and potential effects on earthworms remain unknown. Seven earthworm species belonging to three ecological groupings, with different pigmentation and burrowing behaviour, were tagged using different coloured VIE. External inspection after two days, one week and 1, 10 and 27 months were followed by preservation, dissection and internal inspection. Tags could be seen in living specimens to 27 months, and dissection revealed that in most cases they were lodged in the coelomic cavity, held in place by septa. However, over longer time periods (more than two years), the chlorogogenous tissue tended to bind to the tags and made external observation increasingly diffi cult. Migration of the VIE material towards the posterior of the earthworm and potential loss of the tag were only observed on rare occasions, and a recovery rate in excess of 98% was recorded. By introducing a reasonable amount of VIE into segments, just after the clitellum, this technique can become a valuable tool in earthworm ecology and life history studies, particularly in short-medium term laboratory and fi eld experiments.