J F Ponge

Succession of fungi and fauna during decomposition of needles in a small area of Scots pine litter

Keywords: decomposition, microscopy, pine, soil animals, soil fungi

During micromorphological investigations on Scots pine litter, several decomposition stages have been recognized on fallen pine needles, each being associated with the activity of animal and microbial organisms, both. To well-known fungal successions that have been so far described by mycologists we must add succession of animal groups such as nematodes, amoebae, enchytraeids, sciarid larvae, oribatid mites and earthworms. A bacterial development was observed in the L 2 layer, following penetration by microfauna (nematodes, amoebae).

After that stage pine needles were actively tunnelled by enchytraeids, sciarid larvae and oribatid mites and at the same time were nibbled on by epigeic earthworms (L 2 and F 1 layers). When the fine root system of pine developed through accumulated old needles (F 1 layer), mycorrhizal fungi penetrated the needles and seemed to impede any further bacterial development. Pine foliar tissues were progressively incorporated into the fecal material of earthworms and other members of the soil fauna. A more realistic scheme was suggested for plant litter decomposition in moder humus.

Introduction

Fungal successions during foliage senescence and decomposition have been extensively studied by plant and soil mycologists, especially on pine needles [START_REF] Black | Colonization of Scots pine litter by soil fungi[END_REF][START_REF] Hayes | Studies on the decomposition of coniferous leaf litter. II. Changes in external features and succession of microfungi[END_REF][START_REF] Kendrick | Biological aspects of the decay of Pinus silvestris leaf litter[END_REF]Mitchell and Millar, 1978;Mitchell et al., 1978;[START_REF] Rack | Über Sukzession und pathogene Eigenschaften Kiefernnadeln bewohnender Pilze[END_REF][START_REF] Soma | Ecological studies of soil organisms with references to the decomposition of pine needles. I. Soil macrofaunal and mycofloral surveys in coastal pine plantations[END_REF][START_REF] Watson | Fungal succession on loblolly pine and upland harwood foliage and litter in North Mississipi[END_REF], but similar observations on animals were very scarce [START_REF] Burges | The decomposition of organic matter in the soil[END_REF][START_REF] Styles | Decomposition of Pinus radiata litter on the forest floor. II. Changes in microfauna population[END_REF]. In addition to pines, other coniferous trees have been investigated and strong resemblances may be observed with silver fir (Gourbière, 1981;[START_REF] Gourbière | Champignons des aiguilles de sapin (Abies alba Mill.). VIII. Observation directe des microflores[END_REF][START_REF] Gourbière | Champignons des aiguilles de sapin (Abies alba Mill.). IX. Microflores internes[END_REF]1988;Gourbière and Pépin, 1983). The role of soil fauna has been largely underestimated except when micromorphology was used to follow decomposition processes, as was the case with Douglas fir [START_REF] Bal | Morphological investigation in two moder-humus profiles and the role of the soil fauna in their genesis[END_REF]. When faunal results are discarded, which are in fact difficult to interpret in terms of successions, the commonly admitted sequential pattern for pine needles fungi may be summarized as shown by Table 1. The group of mycorrhizal fungi was only mentioned by [START_REF] Soma | Ecological studies of soil organisms with references to the decomposition of pine needles. I. Soil macrofaunal and mycofloral surveys in coastal pine plantations[END_REF], but their role in the late colonization of pine needles has been fully recognized by [START_REF] Ponge | Etude écologique d'un humus forestier par l'observation d'un petit volume. III. La couche F 1 d'un moder sous Pinus sylvestris[END_REF]1990).

The observations presented here are part of a morphological investigation which was carried out on a small area of pine litter in a temperate forest. Several papers have been already published [START_REF] Ponge | Etude écologique d'un humus forestier par l'observation d'un petit volume, premiers résultats. I. La couche L 1 d'un moder sous pin sylvestre[END_REF]1985a, b;1988;1990;[START_REF] Ponge | Food resources and diets of soil animals in a small area of Scots pine litter[END_REF]. In the present paper, animals and micro-organisms that colonize decaying pine needles are described and the way they succeed each other was studied through comparison of superposed litter layers.

Material and methods

A small area (ca. 5 x 5 cm) of moder humus was sampled on 11.8.81 in a 35-year-old Pinus sylvestris L. plantation (Orleans forest, France) and microstratified in the field into L 1 (brown needles), L 2 (black needles), F 1 (broken needles + pine roots and widespread mycelial mat) and other layers not investigated here. Fixation of each layer was made immediately, before any transport, in 95% ethanol. A more complete description of the stand has been previously given [START_REF] Ponge | Etude écologique d'un humus forestier par l'observation d'un petit volume, premiers résultats. I. La couche L 1 d'un moder sous pin sylvestre[END_REF].

Observations were made under a light microscope at 400 x magnification after sorting the plant and animal material under a dissecting microscope. Pine needles and other plant fragments were sectioned to 7.5-µm thickness (microtome Stiassnie of Minot type with stainless steel knife, after embedding in paraffin wax) and mounted in chloral-lactophenol (25 cm 3 lactic acid + 50 g chloral hydrate + 25 cm 3 phenol). Phase contrast allowed one to discern between dead and living cells at the time of fixation [START_REF] Frankland | Importance of phase-contrast microscopy for estimation of total fungal biomass by the agarfilm technique[END_REF]: presence of cytoplasm was easily discernible by the opacity of cell contents, provided the fungal walls were not melanized.

When necessary (e.g. for dematiaceous fungi), methyl blue was used in lactophenol as a staining agent for cell contents. It helped to observe living fungi and bacteria. Animals were either dissected (oribatid mites) or observed after clearing (other groups) in order to analyse their gut contents. Faecal pellets were embedded and cut like plant material. Recognition of the fragments by external characteristics before cutting them provided a more reliable interpretation of morphological features than direct soil sectioning. The nature of the mycelial mat and its connection to the fungus mantle of mycorrhizae and to the internal colonizers of plant debris and cadavers were also ascertained by direct observation at a low magnification.

Fungal colonizers were identified by morphological characteristics directly observed on the fixed material, without any cultivation. See [START_REF] Ponge | Etude écologique d'un humus forestier par l'observation d'un petit volume, premiers résultats. I. La couche L 1 d'un moder sous pin sylvestre[END_REF]1985a;1988;1990) for further details on the anatomical characteristics that were used for due identification of fungi. Nomenclature for plant anatomy was taken from [START_REF] Esau | Plant Anatomy[END_REF].

Results

Stage I: primary fungal colonizers

Fungal colonization may be easily revealed by the fruiting bodies of Lophodermium pinastri, Ceuthospora pinastri (Fig. 1) and Lophodermella sp. Of these three fungi, L. pinastri was the most commonly encountered.

Several colonies of this ascomycete may occur on the same needle, each one being delimited by two black diaphragms. Figure 1 shows that L. pinastri and C. pinastri may coexist on the same needle, but in distinct colonies separated by the black stromatic lines of the first species which always excludes any other fungus.

Examination of needles collected by a panel several decimeters above the ground surface did not reveal the presence of C. pinastri. Thus we may think that fruiting of this fungus took place after the needles had fallen on the forest floor, which was not the case for the two other fungi. It nevertheless belongs to the same decay stage, since we never observed it succeeding to the two other fungi. The internal development of these three fungi was similar, with hyphae travelling through free spaces in the mesophyll tissues, without any penetration of plant cells. Mesophyll cells appeared to be collapsed, with brown walls. In the stele, phloem tissue was brown and collapsed, but xylem tracheids and vascular fibers kept intact their structure and clarity (Fig. 2). Thus these fungi did not attack lignified tissues. Cellulose walls did not disappear, but seemed to be slightly transformed (distinct browning).

Many needles, light brown in colour and seemingly free of any fungal contamination, when sectioned, proved to be invaded by a hyaline fungus. No fruiting bodies were visible. The most intense development was observed in the substomatal chambers, probably at the expense of the obturating cell, which had disappeared.

Nevertheless, hyphae of this fungus were present in all parts of the sections. Living cells were penetrated and their cytoplasm had disappeared, being replaced by fungal mycelium. No penetration was observed in tracheids, fibres, nor in the epidermal cells, but mesophyll, endodermis, phloem and stele parenchym cells were penetrated.

A distinct browning was visible in all cellulosic walls, but lignified walls remained transparent (Fig. 3). Thus, contrary to the aforementioned three other species, this fungus had an internal development that affected cytoplasm (except lignified epidermal cells) and to a slight extent all cellulose walls. Since this fungus did not sporulate in the observed needles, no attempt was made to identify it, but it shares similar features with the white "stomata" category designated by [START_REF] Hayes | Studies on the decomposition of coniferous leaf litter. II. Changes in external features and succession of microfungi[END_REF] for some freshly fallen pine needles.

Stage II: secondary fungal colonizers

Verticicladium trifidum was the most important fungus in the decomposition of pi ne needles. It has been encountered in a living state from the most superficial layer (L 1 ) to the F 1 layer, suggesting that its persistence in each colonized needle probably exceeded one year (see discussion). This fungus may be easily identified through its typical conidiophores protruding from stomatal apertures. V. trifidum commonly acted as a secondary colonizer (after the aforementioned fungi), but several needles in the L 1 layer proved to have been colonized for the first time by this fungus. This was ascertained by the absence of any other fungal species and for some of them by the presence of starch grains in the mesophyll and transfusion tissue parenchyma cells. By comparing many needles invaded by V. trifidum in the three layers investigated, several stages may be recognized in its internal development. Melanized hyphae penetrated through stomatal apertures and developed a stroma that filled the front cavity above the guard cells. This stage was observed on needles colonized by L. pinastri and may be considered as a quiescent period for V. trifidum, whose internal development may take place once the former species has senesced. Coincident development of these two fungi was never observed in sectioned needles. The next step was the penetration of all needle tissues by hyaline hyphae. These were about 5 µm thick and had relatively thick walls that were easily visible in cross sections. They seemed to invade preferentially elongated cells like xylem tracheids, and bundles of hyphae were observed filling resin ducts (Fig. 4). Hyaline hyphae were also present inside stomatal guard cells. Thus, at this stage, melanized stromata were restricted to the front cavity of stomatal apertures. A tangential section through the lower part of stomata showed that the outer walls of guard cells began to be incrusted by a melanin-like substance. This observation deserves attention, because it is a starting point for needle blackening. No action on cell walls (whether they were made of cellulose or lingo-cellulose) was observed at this stage. Blackening of the needles occurred during the following step. On sections it may be seen that this was due to impregnation of the hypodermal basis by melanin-like substances (Fig. 5). Melanization may reach part of the mesophyll columns (Fig. 5). Ligno-cellulose cell walls were disrupted. This was especially true for transfusion tissue, where in some parts pit borders were the only tracheid remnants that were still recognizable. Hyaline hyphae may give rise to stromatic structures in the zones where pine tracheids had completely disappeared. Xylem tracheids seemed to be more resistant, or penetration may have been delayed as compared to transfusion tracheids: their walls seemed to have been reduced in thickness, but they were still perceptible. In the xylem tissue, bordered pits were the commonest route for the fungus passing from one tracheid to another.

Marasmius androsaceus is a basidiomycete known for its typical brown, flattened rhizomorphs (Fig. 6) that commonly enter decaying needles of several conifer species. Emergence of fruit bodies from the needles was not observed in our sample, but the common appearance of this fungus on decaying coniferous litter was taken as presumptive evidence of its identification (Gourbière, personal communication). Mesophyll cells were replaced by a pseudo-parenchymatous fungal tissue, but no lysis was observed in the ligno-cellulosic tracheids.

The development of this fungus was conspicuous only in the L 2 layer and it seemed to be active only during win ter months.

The mycorrhizal ascomycete Cenococcum geophilum Fr. [= C. graniforme (Sow.) Ferd. et Winge] (ascribed to this group by [START_REF] Trappe | Mycorrhiza-forming ascomycetes[END_REF] for its resemblance with Elaphomyces spp.) was observed to have a well developed mycelium inside some needles in the F 1 layer (Fig. 7). These were not colonized by V. trifidum.

Thus we may hypothesize that V. trifidum was inhibitory to C. geophilum and that the latter could not act as a tertiary colonizer. C. geophilum showed a preference for growing between the hypodermis and the mesophyll.

The fact that hyphae of this fungus did not possess any cell content and showed some signs of autolysis indicated that colonization had probably occurred in the previous autumn.

Stage III: ingestion and penetration by fauna

After the stages of fungal decomposition, all needles appeared to have been attacked by soil animals. Fauna may penetrate inside pine needles (microfauna, mesofauna) or may ingest entire pieces that were compacted into faeces (macrofauna). Action of fauna was associated with a bacterial and algal development which was never observed in the primary and secondary fungal stages.

Presence of bacteria (visible through staining with methyl blue) was observed only after needles had been broken up or nibbled on by animals. In several cases, bacterial development was recorded without any penetration by an animal species, but these needles always showed some signs of a faunal attack. In a first stage, bacteria were observed to develop at the expense of fungi, whose hyphae were still recognizable, as was the case with V. trifidum. At a more advanced decay stage, bacterial development was recorded throughout the whole needle and in most cases the only visible plant remains were bordered pits. Bacteria were accompanied by algae, whose chloroplasts were of a deep purple when stained with methyl blue, or by cyanobacteria.

Protozoa were traced by the presence of cysts, but nematode worms and rotifers were also commonly encountered. Bacteria were present too. It seems that these animals were able to penetrate the needles by their own means, since some needles with microfauna did not present signs of attack by larger-sized animals. Thus they could be considered as a specific stage, half-way between the two first fungal stages and the other faunal stages. Nevertheless, penetration by microfauna was commonly associated with nibbling or fragmentation by other animal species. The same situation was encountered with V. trifidum (see above).

Colonization by mesofaunal groups was known to be active at the sampling time, since in addition to recognizable fecal pellets each group was represented by living animals with filled intestines.

Enchytreid worms were the most commonly encountered mesofaunal group in the three layers investigated. Although pine needles were not their only food, at the time of sampling (summer) they were found actively tunnelling pine needles in the L 2 and F 1 layers (Fig. 8). They were not identified at the species level, but probably belonged to the species Cognettia sphagnetorum (Vejdovsky, 1877) because of the observed fragmentative reproduction and high acidity of their environment [START_REF] Healy | Distribution of terrestrial Enchytraeidae in Ireland[END_REF][START_REF] Standen | The production and respiration of an enchytraeid population in blanket bog[END_REF]. Deposition of fecal pellets in the interior of pine needles was observed, but in most cases the faeces were deposited outside the needles. Bacteria proliferated in the fecal material, both inside and outside the needles, except in the F 1 layer where they were replaced by mycorrhizal fungi, in some cases together with filamentous cyanobacteria.

Parenchymatous pine cells were crushed and their shape was no longer recognizable, but lignified cells were preserved. Action of these animals on the cellulosic material was not fully ascertained (in the most obvious cases digestion seemed to be only partial), but they certainly digested hyaline fungal material (Fig. 9). Dematiaceous hyphal walls were preserved (Fig. 9). Galleries of enchytraeid worms at the inside of pine needles were lined with mucus, which was the centre of an intense bacterial development. Much debris, which the animals had been carrying on their tegument, was found lining the galleries, such as pollen grains or Testacean cysts.

Oribatid mites that were tunnelling through pine needles belonged to the two closely related families Phthiracaridae [Rhysotritia duplicata (Michael, 1880)] and Euphthiracaridae (Phthiracarus spp.). No differences were found between these species concerning the ingested tissues, the fate of the ingested material and the way they deposited their fecal pellets, except that pine needles were an obligate growth medium for Phthiracarus larvae, which was not necessarily the case for Rhysotritia. Thus they will be considered together for describing their role in decomposition processes. Oribatid mites deposited their fecal pellets inside the pine needles (Fig. 10). Due to the high degree of comminution of pine cells after they had been broken up by the buccal apparatus, the ingested tissues were not identifiable in the faeces. There was a fairly good relationship between the size of the animals and the degree of comminution of the plant material. Fecal pellets contained cell wall remains, but there was probably a loss of the crystalline structure of cellulose in the inner part of each pellet: cell walls were observed to become dull brown and amorphous. This phenomenon occurred during passage through the mite intestine, since this transformation could be followed on the same individual between two successive food boli. Microbial development wasnot observed in or on the pellets found inside pine needles. Undoubtedly a choice was made among the different types of fungal colonized needles and it seemed that needles colonized by the mycorrhizal C. geophilum (see above) were preferred to needles colonized by V. trifidum, but no attempt was made to quantify this preference.

Pine needles were also ingested by larvae belonging to the Sciaridae family. They were observed tunnelling the needles in a fashion similar to enchytreid worms, i.e. most fecal material was deposited on the outside. Examination of the faeces gave only evidence of resistant pine structures, such as guard cells.

Nevertheless, intestinal guts were filled with pine cells in the process of being dissolved, bordered pits remaining slightly visible, which proved that digestion of ligno-cellulose occurred, at least with the thin-walled transfusion tissue. No microbial development was recorded in the studied faeces, except cyanobacteria at the periphery.

Macrofaunal activity was restricted to the F 1 layer. This feature was not shared by mesofaunal species, which start their activity in the L 1 layer, as was shown above.

Lumbricid worms were the most active macrofaunal group in the studied layers, judged by accumulation of their faeces in the F 1 layer (Fig. 11). They belonged probably to the epigeic species Dendrobaena octaedra (Savigny, 1926) since it was the only one recorded on this site (Bouché, unpublished data). Pine needle tissues were ingested, but they were always mixed with other material (fungi, faeces, pollen grains, etc.). The degree of comminution of pine material was high, although not comparable to that by oribatid mites, due to transit in the gizzard. No transformations seemed to occur in the cell walls. Microbial development was feeble and restricted to some micro-sites or to the periphery. Earthworm faeces were in most cases penetrated by the brown hyphae of the mycorrhizal fungus C. geophilum (Fig. 12).

Two other types of macrofauna faeces were recorded, but they were far fewer in number than earthworm faeces. Their connection to identified species or group of species was highly hypothetical, thus they will be referred to as type A and type B [START_REF] Ponge | Etude écologique d'un humus forestier par l'observation d'un petit volume. III. La couche F 1 d'un moder sous Pinus sylvestris[END_REF]. Type A faeces were tentatively ascribed to slugs (Fig. 13). They were made of needle pieces that had been rejected without any change in their appearance when observed under a dissecting microscope. Examination at a higher magnification under a light microscope proved that pine tissues were intact, even the most delicate ones. Fungal material was present in great abundance inside of ingested needles, attesting that they had been consumed in the litter, not in the tree foliage. No cytoplasm remains were recorded, thus fungal cytoplasm was probably the only food source for these animals. Microbial colonization of type A faeces was restricted to some micro-sites. Similar features were observed in type B faeces, although the ingested needles had been broken into smaller pieces (Fig. 14). They were tentatively ascribed to woodlice [START_REF] Ponge | Etude écologique d'un humus forestier par l'observation d'un petit volume. III. La couche F 1 d'un moder sous Pinus sylvestris[END_REF]. They seemed not to be a good medium for microbial development, algae or bacteria being restricted to micro-sites as in the previous case. Nevertheless they had been penetrated by mesofaunal groups such as oribatid mites, which deposited their fecal pellets in small cavities. The age of some of the type B faeces was ascertained by autolysis of bacterial colonies, the pine material remaining intact.

Penetration by mycorrhizal fungi was not recorded in type A and B faeces, contrary to earthworm faeces.

Stage IV: penetration by mycorrhizal fungi

Faunal activity led to the presence of holes in all pine needles in the F 1 layer, thus allowing the dominant fungi to enter the pine material. Penetration by C. geophilum was recorded above, but with mycelial growth quite distinct from that of fungi growing freely in open space. In the present case, C. geophilum acted as a saprobic species.

After the needles had been tunnelled by mesofaunal species, the aerial form of the two main mycorrhizal fungi present, namely C. geophilum and the hyaline basidiomycete Hyphodontia sp., developed inside the hollow needles without any change in their anatomical features.

Discussion

The proposed scheme for decomposition of pine needles has no universal value, since it was derived from a microscale study strongly limited both in time and space. Nevertheless, comparison with information in the literature on the same subject leads to the conclusion that the observations on the first stages of fungal decomposition were in accordance with those of more comprehensive studies, thus providing credibility to the present results on later stages which were not studied earlier.

L. pinastri, C. pinastri and Lophodermella spp. (also called Hypoderma and Hypodermella in older literature) are widespread pine-specific fungi which have been recorded frequently on living or senescent pine needles [START_REF] Gremmen | Microfungi decomposing organic remains of pines[END_REF][START_REF] Gremmen | Fungi colonizing living and dead tissue of Pinus sylvestris and P. nigra[END_REF][START_REF] Hayes | Studies on the decomposition of coniferous leaf litter. II. Changes in external features and succession of microfungi[END_REF][START_REF] Kendrick | Biological aspects of the decay of Pinus silvestris leaf litter[END_REF][START_REF] Minter | Four species of Lophodermium on Pinus sylvestris[END_REF][START_REF] Minter | Lophodermium on pines[END_REF]Mitchell and Millar, 1978;[START_REF] Mitchell | Studies on decomposition of Scots pine needles[END_REF][START_REF] Mitchell | The biology of Lophodermella conjuncta Darker on Corsican pine needles[END_REF][START_REF] Rack | Über Sukzession und pathogene Eigenschaften Kiefernnadeln bewohnender Pilze[END_REF][START_REF] Soma | Ecological studies of soil organisms with references to the decomposition of pine needles. I. Soil macrofaunal and mycofloral surveys in coastal pine plantations[END_REF]. These fungi were assigned to one decomposition stage (stage 1), due to uniformity in the mode of colonization (extra-cellular hyphae in the mesophyll) and to coexistence of these species in the same superficial layer (needles shed a few months before sampling date).

V. trifidum was also abundantly recorded as a secondary colonizer specific to fallen pine needles [START_REF] Black | Colonization of Scots pine litter by soil fungi[END_REF][START_REF] Burges | The decomposition of organic matter in the soil[END_REF][START_REF] Gremmen | Microfungi decomposing organic remains of pines[END_REF][START_REF] Gremmen | A contribution to the mycoflora of pine forests in the Netherlands[END_REF][START_REF] Gremmen | Fungi colonizing living and dead tissue of Pinus sylvestris and P. nigra[END_REF][START_REF] Hayes | Studies on the decomposition of coniferous leaf litter. II. Changes in external features and succession of microfungi[END_REF][START_REF] Hughes | Studies on micro-fungi. IX. Calcarisporium, Verticicladium, and Hansfordia[END_REF][START_REF] Kendrick | Biological aspects of the decay of Pinus silvestris leaf litter[END_REF][START_REF] Mitchell | Studies on decomposition of Scots pine needles[END_REF]. Observations on the internal development of V. trifidum were scarce, apart from formation of black stromata in the sub-stomatic chambers, from which the conidiophores emerged.

Nevertheless, [START_REF] Kendrick | Biological aspects of the decay of Pinus silvestris leaf litter[END_REF] indicated that internal tissues were infected by hyaline hyphae, dark hyphae being located in the peripheral zone, and that a distinct browning of the host epidermis and hypodermis cell walls occurred under the influence of this fungus. No reference was made in the literature to the action of V. trifidum upon ligno-cellulose (transfusion tracheids) and the formation of scattered microstromata in later stages of development.

M. androsaceus was known as a non-specific internal colonizer of coniferous needles [START_REF] Burges | The decomposition of organic matter in the soil[END_REF][START_REF] Gourbière | Microscopie de la mycoflore des aiguilles de sapin (Abies alba). III. Marasmius androsaceus[END_REF][START_REF] Gourbière | Décomposition des aiguilles d'Abies alba: hétérogénéité du substrat et de la mycoflore, rôle de Marasmius androsaceus[END_REF][START_REF] Mitchell | Studies on decomposition of Scots pine needles[END_REF][START_REF] Newell | Interaction between two decomposer basidiomycetes and a Collembolan under Sitka spruce: Distribution, abundance and selective grazing[END_REF][START_REF] Soma | Ecological studies of soil organisms with references to the decomposition of pine needles. I. Soil macrofaunal and mycofloral surveys in coastal pine plantations[END_REF]. Deterioration of internal tissues was known to be the action of a white-rot fungus, i.e. ligno-cellulolytic. Accordingly, a nearly complete disappearance of the mesophyll tissues was observed in the zone where the fungus had penetrated, but no lytic action towards tracheids was observed. Colonization was restricted to a small zone near the point of penetration. Thus, the impact of M. androsaceus was less drastic than that of V. trifidum, which is not in accordance with the weight losses commonly attributed to M. androsaceus [START_REF] Gourbière | Décomposition des aiguilles d'Abies alba: hétérogénéité du substrat et de la mycoflore, rôle de Marasmius androsaceus[END_REF]. This discrepancy might arise from the fact that in the present sample M. androsaceus seemed to have been active just during a short cold period (winter months) inside pine needles.

The aforementioned fungal successions were already well described in pine species and they were analogous to what was observed on other coniferous genera (Gourbière, 1981;and personal observations).

In contrast, some of the present observations are not supported by the literature. C. geophilum was found at the same decay stage as V. trifidum and M. androsaceus. C. geophilum was surprisingly absent from reports of [START_REF] Kendrick | Biological aspects of the decay of Pinus silvestris leaf litter[END_REF] and others on fungal decomposition of pine needles, probably due to lack of appropriate cultural methods. These workers used a combination of classical isolation techniques and direct observations on the needles in damp chambers. Using this approach, the recovery of mycorrhizal fungi was certainly impossible.

Colonization of pine needles by fauna has been studied by a few authors. [START_REF] Styles | Decomposition of Pinus radiata litter on the forest floor. II. Changes in microfauna population[END_REF] placed pine needles in nylon-mesh bags in forest litter and followed meso-and macrofaunal populations during four years.

Unfortunately, no mention was made of enchytreid worms or microfauna, and the mesh size he used (1 mm) prevented earthworms and other large animals from penetrating the bags. The main similarity with the present results was that phthiracarid mites were present only in well decayed needles. This was also supported by the cultural observations of [START_REF] Hayes | Studies on the feeding preferences of some phthiracarid mites (Acari: Oribatidae)[END_REF] on sever al phthiracarid species. Other studies on the role of soil animals in pine needle decomposition [START_REF] Berg | Influence of soil animals on decomposition of Scots pine needle litter. In Structure and Function of Northern Coniferous Forests: An Ecosystem Study[END_REF][START_REF] Elliott | The role of millipedes in the decomposition of Pinus radiata litter in the Australian Capital Territory[END_REF][START_REF] Hartenstein | Soil Oribatei. VII. Decomposition of conifer needles and deciduous leaf petioles by Steganacarus diaphanum (Acarina: Phthiracaridae)[END_REF][START_REF] Kowal | The ingestion rates of micro-arthropods in pine mor, estimated with radioactive calcium[END_REF][START_REF] Lundkvist | The influence of soil fauna on decomposition of pine needle litter: A field experiment[END_REF][START_REF] Metz | Acarina associated with decomposing forest litter in the North Carolina Piedmont[END_REF][START_REF] Soma | Ecological studies of soil organisms with references to the decomposition of pine needles. II. Litter feeding and breakdown by the woodlouse, Porcellio scaber[END_REF]) are difficult to discuss in this respect, since they were not conducted for an assessment of the impact of soil fauna upon decomposition processes. Direct relevance to decomposition processes could be found in laboratory experiments with enchytreid worms and spruce litter or humus [START_REF] Abrahamsen | Influence of Cognettia sphagnetorum (Oligochaeta: Enchytraeidae) on nitrogen mineralization in homogenized mor humus[END_REF][START_REF] Griffiths | Enhanced nutrient mineralization and leaching from decomposing Sitka spruce litter enchytraied worms[END_REF]. Both these studies and many others on different substrates and animal groups gave evidence of an increased net mineralization rate for nitrogen, but they differed in their answers to the question: is mineral nitrogen (or near-mineral substances such as urea) produced directly by the animals or indirectly, through their action upon microorganisms? The question seems unsolved at the present time, and the matter probably needs more refined methods to be assessed. Another study made with the same material [START_REF] Ponge | Food resources and diets of soil animals in a small area of Scots pine litter[END_REF] establishes a direct influence of some soil animals upon plant material through digestion processes.

Legends of figures

Fig. 1 .

 1 Fig. 1. Infection by Lophodermium pinastri (tip of the needle) and Ceuthospora pinastri (minute black spots). L 1 layer. Bar = 3 mm.

Fig. 2 .

 2 Fig. 2. Infection by Lophodermium pinastri. From right to left: vascular fibres, phloem (note the browning), xylem, transfusion tissue. L 1 layer. Staining with methyl-blue. Bar = 50 µm.

Fig. 3 .

 3 Fig. 3. "White stomata" fungus. Phloem (brown) and xylem (clear). L 1 layer. Staining with methyl-blue. Bar = 50 µm.

Fig. 5 .

 5 Fig. 5. Blackening of hypodermis and outer part of mesophyll rays following invasion by Verticicladium trifidum. From right to left: central cylinder, transfusion tissue, endodermis, mesophyll rays, hypodermis, epidermis. Staining with methyl-blue. L 2 layer. Bar = 50 µm.

Fig. 6 .

 6 Fig. 6. Rhizomorph of Marasmius androsaceus. Phase contrast. L 2 layer. Bar = 50 µm.

Fig. 7 .

 7 Fig. 7. Internal development of Cenococcum geophilum. Two forms: poorly ramified (aerial form) and strongly ramified (internal form). Phase contrast. F 1 layer. Bar = 50 µm.

Fig. 8 .

 8 Fig. 8. Two enchytraeid worms (arrows) penetrating a needle at the time of fixation. F 1 layer. Bar = 2 mm.

Fig. 9 .

 9 Fig. 9. Dematiaceous and hyaline hyphae in the intestine of Cognettia sphagnetorum. Phase contrast. L2 layer. Bar = 50 µm.

Fig. 10 .

 10 Fig. 10. Partially dissected pine needle showing accumulation of phthiracarid mite fecal pellets. F 1 layer. Bar = 3 mm.

Fig. 11 .

 11 Fig. 11. Earthworm fecal pellet. F 1 layer. Bar = 1 mm.

Fig. 12 .

 12 Fig. 12. Cenococcum geophilum hyphae protruding from an earthworm fecal pellet. F 1 layer. Bar = 2 mm.

Fig. 13 .

 13 Fig. 13. Type A faeces. F 1 layer. Bar = 2 mm.

Fig. 14 .

 14 Fig. 14. Type B faeces. Broken pine needles. F 1 layer. Bar 1 mm.

  Fig. 1

Table 1 .

 1 Succession of fungi during decomposition of pine needles Saprophytic senescence fungi Lophodermium pinastri (Schrad.) Chev. Ceuthospora pinastri (Fr.) Hohnel (imperfect stage of Phacidium lacerum Fr.) Lophodermella spp. Litter-decomposing micro-fungi Verticicladium trifidum Preuss (imperfect stage of Desmazierella acicola Lib.) Litter-decomposing basidiomycetes Marasmius androsaceus Fries

		Collybia spp.
	Mycorrhial fungi	unidentified

Acknowledgements

The author is indebted to Prof D A Crossley Jr (Athens, USA) and to Drs P J A Howard and J C Frankland (Merlewood, England) for their help in improving the English.


F Beniamino

J F Ponge

P Arpin

Soil acidification under the crown of oak trees. I. Spatial distribution

Thirty oak trees (Quercus robur L. sensu lato) growing on the same site were selected on the basis of their stem diameter (D bh ≥ 0.625 m) and their effect on soil properties was assessed. Litterfall, old litter accumulation, acidity and buffering capacity of the A 1 horizon were measured at three distances from the trunk (0.4, 1.4 and 2.4 m) and in four directions (N, E, S, W). Results led to the conclusion that on average more acidification and litter accumulation occurred near the trunk base and in the north direction, but this general trend was far from being followed by every tree. The clay content of the soil was inversely related to acidification and litter accumulation. Interrelationships between soil organisms, crown leaching, bark substances and the parent rock are proposed as working hypotheses for the future of this study.

INTRODUCTION

During the last fifteen years soil acidification processes have attracted increased interest due to consequences of industrial pollution over forested areas, especially in North and Central Europe [START_REF] Tamm | Acid precipitation: biological effects in soil and on forest vegetation[END_REF][START_REF] Abrahamsen | Effects of acidic deposition on forest soil and vegetation[END_REF]Wittig et al., 1985;[START_REF] Van Breemen | Acidification and decline of Central European forests[END_REF][START_REF] Kauppi | Acidification of forest soils: model development and application for analyzing impacts of acidic deposition in Europe[END_REF][START_REF] Wittig | Acidification phenomena in beech (Fagus sylvatica) forests of Europe[END_REF][START_REF] Falkengren-Grerup | Soil acidification and vegetation changes in deciduous forest in southern Sweden[END_REF], 1987;[START_REF] Hallbäcken | Changes in soil acidity from 1927 to 1982-1984 in a forest area of South-West Sweden[END_REF][START_REF] Tyler | Probable effects of soil acidification and nitrogen deposition on the floristic composition of oak (Quercus robur L.) forest[END_REF]. Acidification markedly affects the area near the trunk base of trees, as has been assessed by soil pH and vegetation data [START_REF] Zinke | The pattern of influence of individual forest trees on soil properties[END_REF][START_REF] Lane | Comparison of pH of pine stem, bark, roots, and adjacent soil[END_REF][START_REF] Wittig | Acid indicators around the trunk base of Fagus sylvatica in limestone and loess beechwoods: distribution pattern and phytosociological problems[END_REF][START_REF] Cloutier | Microdistribution des espèces végétales au pied des troncs d'Acer saccharum dans une érablière du sud du Québec[END_REF]Riha et al., 1986a,b;[START_REF] Wittig | Acidification phenomena in beech (Fagus sylvatica) forests of Europe[END_REF] and study of the mycorrhizal types [START_REF] Kumpfer | Effects of stemflow on the mycorrhiza of beech (Fagus sylvaticus L.)[END_REF]. This phenomenon has been mainly attributed to stemflow, but bark falling near the trunk has been also implicated [START_REF] Zinke | The pattern of influence of individual forest trees on soil properties[END_REF]. The acidic nature of water running down the trunk was established by several studies on oak [START_REF] Carlisle | The nutrient content of tree stem flow and ground flora litter and leachates in a sessile oak (Quercus petraea) woodland[END_REF] and other trees [START_REF] Mina | Influence of stemflow on soil[END_REF]. Given its richness in cations, it may be thought that the acidity of stemflow is mostly due to the charge of water-soluble phenolic substances leached from bark and their chelating properties [START_REF] Updegraff | Microbial utilization of Pinus radiata bark[END_REF][START_REF] Olsson | Properties and decomposition of bark[END_REF]: [START_REF] Mina | Influence of stemflow on soil[END_REF] pointed out that rough bark gives a more acidic stemflow than does smooth bark. [START_REF] Wittig | Acidification phenomena in beech (Fagus sylvatica) forests of Europe[END_REF], in view of the large amount of water collected by the trunk, especially on trees where branches fork at an acute angle [START_REF] Aussenac | Interception des précipitations par le couvert forestier[END_REF][START_REF] Aussenac | Action du couvert forestier sur la distribution au sol des précipitations[END_REF], invoked pollution by rain and mist in order to explain the observed acidification of the stemflow area (ground area influenced by stemflow). He derived this hypothesis from a comparison of the number of acid indicators (plant species) in the stemflow area through several beech forests of Central (polluted) and South (less polluted) Europe. Doubts can be raised against this contention because of the different nature of humus in warmer countries, where organic matter rarely accumulates on the top of soil. In addition, such 'acid' indicators as Deschampsia flexuosa are in fact resistant to phenolic substances [START_REF] Kuiters | Effects of phenolic acids on growth, mineral composition and chlorophyll content of some herbaceous woodland species[END_REF].

In a previous study [START_REF] Arpin | Utilisation des nématodes Mononchida et des Collemboles pour caractériser des phénomènes pédobiologiques[END_REF] a strong modification of the humus type, with contingent acidification, was observed under the crown of an old oak tree. In the present study, this effect was investigated in order to verify its generality and derive some working hypotheses concerning the causes involved.

In this paper, soil acidity and litter accumulation will be described. Complete data have been presented in a more extensive paper [START_REF] Beniamino | Etude de l'acidification sous les chênes de réserve dans un T.S.F. de la forêt domaniale de Sénart[END_REF]. Experimental studies will be reported in a subsequent contribution.

SITE DESCRIPTION

The study area has been described previously (Arpin et al., 1984, stations 3 and 4). It is a coppice with standards (full-grown trees) approximately 150 years old, growing on a gentle slope facing the river Seine (state forest of Senart, 30 km south of Paris). Standards are sessile oaks [Quereus petraea (Mattuschka) Liebl.] except one single pedunculate oak [Q. robur L.], but hybridation, indicated by thorough examination of the leaves, seems to be very frequent. Some trees are isolated but generally the crowns join each other. The coppice comprises lime (Tilia cordata Mill.) and hornbeam (Carpinus betulus L. ). Their development is hampered by competition for light with oak canopies, except in some places. The soil is a loam to clay loam brown leached soil (luvi-to cambisol under the FAO classification; [START_REF] Duchaufour | Pédologie. I. Pédogénèse et Classification[END_REF], with boulders up to the ground surface.

Usually the humus is an acid mull, with a pH of the A 1 horizon ranging from 4 to 5, and we noticed an obvious mole activity, whose importance in mull formation has been stressed by [START_REF] Bornebusch | The fauna of forest soil[END_REF]. The ground vegetation is mainly of bramble (Rubus sp.), ivy (Hedera helix L.), dog's mercury (Mereurialis perennis), yellow dead-nettle (Galeobdolon luteum Huds.), oak seedlings and abundant spring flowering species, such as squill [Scilla bifolia (L.)], bluebell [Endymion non-scriptus (L.) Garcke] and wood-anemone (Anemone nemorosa L.).

More detailed description of the site has been presented elsewhere [START_REF] Beniamino | Etude de l'acidification sous les chênes de réserve dans un T.S.F. de la forêt domaniale de Sénart[END_REF].

METHODS

Thirty trees were randomly selected among 74 growing in the same compartment, each with ~0.625 m breastheight diameter. Four directions corresponding to the cardinal points were sampled in order to assess the possible influence of winds on litter distribution and influence of light and rains on soil properties. Since it was quite impossible to compare the soil under an oak crown to other places not influenced by trees, it was arbitrarily decided to sample at three distances from the trunk base, i.e. 0.4, 1.4 and 2.4 m. Combination of the two factors under study (direction and distance from the tree trunk base) gave 30  4  3 = 360 sampling points.

Litter fall and accumulation were measured at each sampling point just after the main fall (end November 1988). A stainless steel 15 cm  cylinder with chamfered edge was forced into the soil. Fresh litter (L 1 layer) and accumulated organic matter (L 2 to H layers) were separately collected, air-dried then weighed.

Fresh litter was expressed as total litter, and in addition subdivided into oak, beech, lime, hornbeam, sycamore, bramble leaves and miscellaneous (wood, bark, acorns, etc.). The A 1 horizon was sampled down to 10 cm, then air-dried, sieved to 2 mm and preserved for acidity and buffering capacity measurements.

Acidity of the soil solution was measured in a l:2 soil:water suspension (50 g soil + 100 g deionized water) after the mixture had settled for 24 h. Measurement of pH was made with a glass electrode (with a KCI electrode included as a reference) in constant agitation. Titration was made with a 1N NaOH solution up to pH 7.

Immediate titration indicated the more easily available acidity. Titration after 1 and 24 h revealed more stable acidity forms. The buffering capacity was expressed as the quantity of NaOH necessary to rise the pH of one half-unit above the immediate reading. All these measurements were made on the same soil suspension.

Statistical analyses were performed using three-way analyses of variance [START_REF] Sokal | Biometry. The Principles and Practice of Statistics in Biological Research[END_REF][START_REF] Dagnélie | Théorie et méthodes statistiques[END_REF]. Given the strong heterogeneity between trees, they were considered as blocks. Combinations of the three distances and the four orientations were the treatments applied to each block. The mixed model used was:

X ijk =µ+ i + j +C k + ijk
where X was the observed value, µ was the mean,  and P were fixed treatment effects (distance and orientation), C the random block effect and  the residual. Interaction terms have been omitted. Data were transformed whenever necessary in order to normalize the distribution of the residues (homoscedasticity of the data), thus insuring additivity of the effects. Multiple comparisons were made between means using Newman-Keuls a posterior tests. Product-moment correlation coefficients between variables were calculated with trees as pairs. Ranks were used instead of measured values, in order to include some variates that did not meet assumption of analysis of variance, particularly variates with too many null values (leaf components other than oak). Measurements that were not made at each sampling plot but pooled for each tree, such as data (%) on soil particle size, were included only in correlation studies (Spearman rank correlation coefficient: [START_REF] Sokal | Biometry. The Principles and Practice of Statistics in Biological Research[END_REF]. This was also the case for tree height, stem diameter, and degree of crown competition. Simple correlation was used as a tool for measuring the degree of co-variation between two variables. Testing the null hypothesis was made using statistical tables [START_REF] Fisher | Statistical Tables for Biological, Agricultural and Medical Research[END_REF][START_REF] Rohlf | Statistical Tables[END_REF] but the authors are conscious of the fact that some significant correlation coefficients might have been produced by chance when ca1culated on a great number of couples. The threshold of significance of the tests, both for variance analysis and correlation studies, was fixed at 1 %.

RESULTS

Variance analysis

Table 1 summarizes the results of the analyses of variance which were run on ten variables, after suitable transformations. The more conspicuous phenomena are acidification and accumulation of organic matter near the tree trunk base. The most significant trends are exhibited by old litter areal weight (1.5 between 1.4 and 0.4 m, 2.4 between 2.4 and 0.4 m), immediate titration (1.4 between 1.4 and 0.4 m), and titration after 24 h (1.7 between 1.4 and 0.4 m). All these trends are significant at the 0.0001 level. Although this variation is significant only at the 0.01 level, pH is lowered by 0.17 unit (proton concentration magnified by 1.5) between 1.4 and 0.4 m. Thus all these trends are of the same order of magnitude. Total fresh litter and miscellaneous litter do not exhibit any significant trend, but this is not true of oak litter, with a slight but marked increase in fall between 1.4 and 0.4 m (1.14).

Interaction between the two treatment levels (distance and orientation) was never significant even at the 5% level, thus enabling the testing of these two treatment levels separately. Variation between trees was always highly significant (P<0.0001), indicating that large discrepancies exist between them in their response to distance and orientation. Table 2 summarizes the variation between trees.

Correlation

It was necessary to know whether or not the trees around which organic matter was accumulating and those around which soil acidification was occurring were the same. This was not indicated by separate analyses of variance and so data were pooled for each tree (Table 2) and compared from one tree to another. In addition, gradients from 1.4 to 0.4 m for the most significant variates were calculated by subtracting values at 1.4 m from the values measured at 0.4 m (pooled for the four directions). The same calculations were performed between north and south values.

Total, miscellaneous and lime litter are correlated (r = 0.85 between total and miscellaneous litter, r = 0.54 between lime and miscellaneous litter). Lime litter and beech litter are correlated with the fine-silt content of the soil but in an opposite way (r = -0.49 and 0.54). In this case we may question the validity of rejecting the null hypothesis, but we cannot discard some possible influence of the substrate on the presence or development of lime and beech understory under the crown of trees. Bramble and hornbeam litters are not correlated with any other measurement.

Oak litter data, when pooled under each tree, do not exhibit any significant correlation, but the difference between 0.4 and 1.4 m is correlated to that for pH of the Al horizon (r = -0.50). This means that the higher oak litter accumulates at the tree trunk base the higher is the corresponding gradient of acidification.

Soil pH and accumulated old litter are negatively correlated at the 0.001 level (r = -0.76). Thus, trees that accumulate organic matter in the upper horizons of the soil are the same under which the A 1 horizon is acid.

Nevertheless, the same relation was not found between decrease of pH between 1.4 and 0.4 m (sample points) and increase of old litter accumulation at the same distance.

Other relationships are quantified by the product-moment correlation, but they concern the different kinds of acidity and their relation to accumulated organic matter. Both pH and old litter areal weight are correlated with immediate titration (r = -0.58 and 0.50 respectively) and titration after 1 h (r = -0.59 and 0.54).

Decrease of soil pH between 1.4 and 0.4 m is negatively correlated with a similar gradient in immediate titration (r = -0.55), titration after 1 h (r = -0.55) and titration after 24 h (r = -0.65). Immediate titration is correlated with titration after 1 h (r = 0.74). Buffering capacity is correlated with pH (r = -0.46), immediate titration (r = 0.90), titration after 1 h (r = 0.70) and accumulation of organic matter (r = 0.53).

Among factors which may influence pH or accumulation of litter, the clay content of the soil, which is correlated with these two variates (r = 0.65 and -0.56 respectively), may be highlighted. This means that the trees growing on a soil containing more clay do not acidify and accumulate organic matter as much as trees growing on a soil poorer in clay. This is confirmed by a significant correlation between old litter accumulation and the coarse sand content of the soil (r = 0.47). Significant correlation between fine-sand content of the soil and titration after 24 h (r = 0.56) may be also related to the same phenomenon. Unlike oak litter, beech litter exhibits a significant correlation with accumulation of organic matter (r = 0.54), but this cannot be taken as a causal relationship, since beech is very sparsely distributed in the stand under study.

Height of the trees and stem diameter are never correlated with the other variates, they are just correlated between themselves (r = 0.57). It must be noticed that these two measurements are not informative either of the age of the trees or of their productivity, since some of the trees grew freely, but some others did not, as ascertained by crown development and height of the main branching.

Differences between north and west in soil acidity and litter accumulation were calculated and compared to the other variates. Opposition between north and west is correlated with the trunk effect (gradient between 1.4 and 0.4 m) for litter accumulation (r = 0.49), indicating that similar factors influence these two phenomena. A similar relation was not evidenced for pH, but it must be noticed that the contrast between the north and the west side of the tree for pH is correlated with the gradient 0.4-1.4 m for titration after 24 h (r = -0.56).

DISCUSSION

Despite of the fact that autumn litterfall is highly variable from one tree to another (from 580 to 807 g/m 2 ), no relation was found between litter accumulation and litter fall (r = 0.08 and 0.04 respectively). The question which arises is whether or not litter is displaced by wind once on the ground. Since no influence of the direction was visible on the data, and oak litterfall is more pronounced near the trunk base (which correspond to the position of the top of the crown) this indicates that redistribution under the influence of winds was probably negligible. Relation between the increase in the part played by oak leaves in litter near the trunk base and increase of acidity in the same area may nevertheless be highlighted. Examination of the data of [START_REF] Ovington | Studies of the development of woodland conditions under different trees. I. Soil pH[END_REF], and our own measurements on oak leaves (L layer) soaked in water, show values around pH 5 in the leaf leachate. This is not very different from the pH of deionized or distilled water (after equilibration with air). More probably, oak leaf litter and oak foliage have effects through the organic acids they produce during crown leaching and decomposition. These are weak proton producers but very active complexing agents, especially aliphatic acids [START_REF] King | The effects of drying and ageing tree leaves on the ability of their aqueous extracts to dissolve ferric oxide[END_REF][START_REF] Bruckert | Influence des composés organiques solubles sur la pédogénèse en milieu acide. I. Etudes de terrain[END_REF].

The relation between stemflow and acidification of the soil around the trunk may be questioned in the case of oak-trees, since most of the rain running down branches does not reach the soil. Stemflow is only 0.62% of the incident rain in an oak stand of similar age and in the same geographic region, with trees branching at a more acute angle [START_REF] Nizinski | Mesures et modélisation de l'interception nette dans une futaie de chênes[END_REF]. In the present study, observations during showers in winter did not indicate any arrival of water to the ground, probably due to the right-angle branching of the trees. The stem is wetted only by rainwater falling directly on the bark, the effect being well-marked when trees are bending down eastwards. Nevertheless, slow diffusion of water-soluble substances from the stump bark and the fall of pieces of bark must be considered. Given the high tannin content of bark [START_REF] Updegraff | Microbial utilization of Pinus radiata bark[END_REF][START_REF] Olsson | Properties and decomposition of bark[END_REF] and pH of the leachate when soaked in water (between 3 and 4 in our own experiments, against 5 in Olsson's experiments ), it is probable that some area around the trunk base is affected by this source of acidity. This does not explain why the north side is more affected than the west side of the tree. We may hypothesize that perhaps some pollutants such as SO 2 whose absorption by foliage is very feeble [START_REF] Lovett | Dry deposition and canopy exchange in a mixed oak forest as determined by analysis of throughfall[END_REF], are responsible for such a situation: pollution, which is not negligible in the Senart forest (indicated by the absence of hairy lichens), comes mainly from true north, this forest being situated 30 km south of Paris.

The role of the clay content of the soil, as an antagonism to the process of acidification, becomes apparent from our data, since a great part of the variation between trees may be explained by this soil component. The role of clay (and iron) as a driving agent in mull formation is well-known [START_REF] Duchaufour | Pédologie. I. Pédogénèse et Classification[END_REF]), but many hypotheses may account for it. Between them, we may highlight the detoxifying role of clay minerals: they precipitate phenolic and aliphatic acids [START_REF] Tan | Principles of Soil Chemistry[END_REF], thus rending the soil solution more suitable to mullforming species.

TABLE 1

 1 , **, *** = significant at the 0.01, 0.001, 0.0001 level. When heterogeneity among treatments is significant, mean values belonging to the same group (Newman-Keuls test) are followed by a common letter.

	Analysis of variance
	Measurement	Distance 40 cm 140cm 240 cm Orientation North East	South West
	Total fresh litter (g/m 2 )	n.s.	608	593	564	n.s.	550	624	593	578
	Oak fresh litter (g/m 2 )	*	371a	326b	318b	n.s.	340	331	342	340
	Fresh litter miscellaneous (g/m 2 )	n.s.	169	185	169	n.s.	144	214	169	169
	Old litter (g/m 2 )	***	3116a	2024b	1288c	*	2450a 2010ab 2296a 1596b
	pH H2O	*	4.41a	4.58b	4.49b	*	4.41a	4.45ab	4.58b	4.58b
	Buffering capacity (meq/ 100 g air-dried soil)	n.s.	0.23	0.21	0.20	n.s.	0.23	0.20	0.21	0.23
	Immediate titration (meq/ 100 g air-dried soil) ***	1.64a	1.26b	1.19b	n.s.	1.48	1.40	1.33	1.33
	Titration 1 h (meq/ 100 g air-dried soil)	n.s.	0.79	0.79	0.79	n.s.	0.89	0.79	0.74	0.84
	Titration 24 h (meq/ 1 00 g air-dried soil)	***	1.00a	0.65b	0.74b	n.s.	0.89	0.70	0.74	0.84
	Titration 1 h=NaOH IN added 1 h after immediate titration to adjust pH to 7.
	Titration 24 h=NaOH IN added 24 h after to adjust pH to 7.
	n.s. = not significant at the 0.01 level.

*

TABLE 2

 2 Pooled data for each of the 30 trees under study (each tree is indicated by a number in top of each column)

	Measurement	13	27	26	21	14	17	18	15	30	20	1	7	6	3	8
	Total fresh litter (g/m 2 )	578	460	496	656	448	707	564	564	672	564	608	624	725	780	448
	Oak fresh litter (g/m 2 )	391	321	310	388	310	400	368	279	453	400	368	252	276	374	266
	Lime fresh litter (g/m 2 )	5	2	3	26	7	11	1	17	7	18	18	24	41	16	15
	Bramble fresh litter (g/m 2 )	5	4	1	3	19	4	15	21	0	10	17	31	6	9	2
	Hornbeam fresh litter (g/m 2 )	0	8	17	3	0	6	1	0	53	1	0	1	1	1	2
	Beech fresh litter (g/m 2 )	3	1	3	17	6	4	8	2	0	1	0	0	0	0	1
	Maple fresh litter (g/m 2 )	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Fresh litter miscellaneous (g/m 2 )	149	107	127	190	103	227	140	196	159	107	131	262	309	317	135
	Old litter (g/m 2 )	7733	5853	4462	4323	4224	3559	3469	2999	2773	2450	2189	1983	1950	1943	1910
	pH H2O	3.76	4.12	4.37	4.33	4.24	4.45	4.08	4.16	4.41	4.58	4.20	4.37	4.20	4.67	4.54
	Buffering capacity (meq/100 g air-dried soil)	0.15	0.39	0.28	0.33	0.23	0.23	0.27	0.18	0.21	0.14	0.33	0.47	0.27	0.20	0.42
	Immediate titration (meq/100 g air-dried soil)	1.25	2.01	1.91	1.64	1.40	1.33	2.12	1.12	1.48	0.89	1.64	2.46	2.01	1.26	2.12
	Titration 1 h (meq/100 g air-dried soil)	0.50	1.40	0.79	1.12	1.33	0.70	1.12	1.33	1.00	0.50	0.64	1.56	1.56	0.74	2.23
	Titration 24 h (meq/100 g air-dried soil)	0.79	2.58	1.33	0.74	0.84	0.74	0.65	0.74	0.79	0.57	1.64	0.79	0.65	1.48	0.74
	Tree height (m)	23.0	25.0	24.0	22.0	22.0	22.5	22.0	22.0	25.0	21.0	22.0	23.0	22.5	22.5	23.0
	Stem diameter (cm)	71	120	79	68	68	64	69	67	82	66	71	65	64	73	82
	Clay (%)	11.4	13.8	31.3	14.4	14.8	18.5	17.6	22.2	16.4	30.2	19.2	21.6	22.1	21.2	21.0
	Fine silt (%)	25.6	23.2	22.1	20.9	30.7	27.1	26.4	25.4	24.4	27.5	14.6	23.1	22.7	21.5	24.3
	Coarse silt (%)	18.0	14.5	1.9	15.4	12.4	18.9	18.9	16.7	17.3	2.3	3.1	18.0	14.2	10.8	16.9
	Fine sand (%)	15.4	21.9	20.3	17.7	15.3	14.0	12.5	13.9	15.9	14.5	21.7	13.2	15.7	17.9	13.6
	Coarse sand (%)	29.6	26.6	24.4	31.7	26.8	21.5	24.6	21.8	26.1	25.6	41.5	24.1	25.3	28.7	24.1
	Measurement	16	5	9	29	4	24	22	11	2	12	23	10	25	19	28
	Total fresh litter (g/m 2 )	656	725	725	861	578	509	593	403	689	363	578	578	425	820	522
	Oak fresh litter (g/m 2 )	437	307	406	297	238	269	391	240	326	242	377	403	334	513	307
	Lime fresh litter (g/m 2 )	23	22	18	38	16	25	7	17	19	16	20	12	8	11	7
	Bramble fresh litter (g/m 2 )	2	8	1	2	34	0	0	11	6	15	1	3	1	7	0
	Hornbeam fresh litter (g/m 2 )	5	1	5	18	3	1	1	8	0	14	2	7	4	16	1
	Beech fresh litter (g/m 2 )	2	0	0	0	0	0	5	0	0	0	2	0	1	0	0
	Maple fresh litter (g/m 2 )	2	2	0	0	1	0	0	0	0	0	0	0	0	0	14
	Fresh litter miscellaneous (g/m 2 )	164	353	262	448	262	164	169	114	300	70	149	127	64	227	140
	Old litter (g/m 2 )	1787	1731	1687	1501	1415	1331	1182	1182	1121	1121	951	914	654	554	482
	pH H2O	4.41	4.67	4.49	4.16	4.88	4.75	4.54	4.58	4.41	4.45	4.75	4.67	5.24	5.52	5.20
	Buffering capacity (meq/100 g air-dried soil)	0.17	0.25	0.21	0.25	0.13	0.21	0.25	0.12	0.13	0.14	0.18	0.15	0.20	0.14	0.17
	Immediate titration (meq/100 g air-dried soil)	1.26	1.82	1.40	2.01	0.74	1.33	1.91	0.79	1.00	1.12	1.40	1.00	0.89	0.65	0.89
	Titration 1 h (meq/100 g air-dried soil)	1.19	1.26	0.43	0.95	0.65	0.57	0.74	0.24	0.79	0.57	0.61	0.61	0.43	0.34	0.43
	Titration 24 h (meq/100 g air-dried soil)	0.61	0.61	0.34	1.12	1.64	0.74	0.74	0.24	1.48	0.46	1.00	0.46	0.43	0.26	0.95
	Tree height (m)	26.5	23.0	28.5	25.0	22.0	24.0	20.0	24.5	22.0	23.0	21.5	25.0	22.0	21.0	31.0
	Stem diameter (cm)	97	90	91	65	67	67	65	66	69	64	63	75	63	67	74
	Clay (%)	24.5	24.4	26.8	19.2	25.8	24.7	24.8	20.9	18.0	24.5	34.8	25.9	37.5	23.0	23.3
	Fine silt (%)	22.6	14.4	22.5	19.4	13.6	20.9	26.4	23.2	19.4	19.4	22.4	20.7	23.1	24.3	23.9
	Coarse silt (%)	18.6	12.9	12.2	19.0	15.0	18.2	13.6	16.3	13.8	19.0	5.3	18.5	2.2	17.4	17.1
	Fine sand (%)	14.0	16.3	13.8	17.7	16.4	12.8	15.8	13.7	15.1	15.4	16.3	14.0	15.2	15.3	15.1
	Coarse sand (%)	20.3	32.1	24.7	24.8	29.1	23.4	19.4	25.9	33.7	21.7	21.3	20.9	22.0	20.0	20.7

ACKNOWLEDGEMENTS

We are very grateful to Dr. Gloria Nombella for her assistance. We also like to thank Prof. Amyan Macfadyen (U.K.) and Prof. Dennis Parkinson (Canada) for careful examination of the manuscript and revision of the English language.