Microbial characteristics of soils depending on the human impact on archaeological sites in the Northern Caucasus

Quaternary International 324 (2014) 162e171

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Microbial characteristics of soils depending on the human impact on archaeological sites in the Northern Caucasus Swetlana Peters a, *, Aleksander V. Borisov b, Sabine Reinhold c, Dmitrij S. Korobov d, Heinrich Thiemeyer a a

Institute of Physical Geography, Goethe-University, Altenhöferallee 1, 60438 Frankfurt am Main, Germany Institute of Physicochemical and Biological Problems of Soil Science, Pushchino, Russia Eurasia-Department, German Archaeological Institute, Berlin, Germany d Institute of Archaeology, Russian Academy of Sciences, Moscow, Russia b c

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 24 December 2013

Anthropogenic impact in prehistoric settlements results in a considerable alteration of soil microbial communities depending on intensity and the character of human activities. This paper present a case study from a Late Bronze Age settlement located in the high-mountain part of the North Caucasus (Russia). The site represents a community, which presumably specialized in intensive livestock herding. Samples from settlement soils anthropogenically affected in the past and unmodified background soils were taken and studied. Of particular interest were divergences in soil microbial communities, expected to indicate different activities and animal presence in the site. The total microbial biomass, their respiratory activity, the biomass of fungal mycelium and the proportion of dark-colored hyphae were determined, as well as the quantitative state of keratinophilic fungi. The microbial characteristics vary considerably within the settlement locations, and contrast sharply with the reference soils exterior to the archaeological site. The cultural layer has higher percentage of active metabolizing microorganisms, whereas the total microbiological biomass is considerably lower than in the unmodified soils from the surroundings. A determining factor to transform the respiratory activity of microorganisms, in both qualitative and quantitative aspects, is the composition of the organic material which has been accumulated in the ground as a result of various human activities in the past. The cultural layers contain microorganisms, which can be reactivated when glucose is added. In the anthropogenically unmodified soils surrounding the prehistoric settlement, in contrast, 97% of the cells cannot be reactivated. Based on the mycological characteristics of the studied cultural layers and unmodified soils, in particular with regard to the total biomass of fungi mycelium, the dark pigmented fungal biomass, and the existence of keratin-decomposing soil fungi, detailed information about activity areas and their specific usage is given. The use of bio-indicators allows not only diagnosing anthropogenic impact in soils as such, but also significantly complements description of cultural layers of activity areas in the settlement, specifying their purpose. The paper presents the microbiological analyses applied and, moreover, discusses the potential of this approach as a non-destructive prospecting method on archaeological sites. Ó 2013 Elsevier Ltd and INQUA. All rights reserved.

1. Introduction Soil studies within archaeological sites have a long history, but most of them are concerned with general aspects of soil chemistry, in particular pH-variation, microelement studies, or

* Corresponding author. E-mail address: [email protected] (S. Peters). 1040-6182/$ e see front matter Ó 2013 Elsevier Ltd and INQUA. All rights reserved. http://dx.doi.org/10.1016/j.quaint.2013.11.020

micromorphology (e.g. Limbrey, 1975; Middleton and Price, 1996; Holliday and Gartner, 2007; Thiemeyer, 2009; Nicosia et al., 2011). In recent years, however, soil studies related to archaeological projects in Russia have introduced a microbiological perspective to the investigation of paleosols and sediments that had been anthropogenically influenced (Demkina et al., 2000; Kashirskaya, 2006; Demkin et al., 2008; Khomutova et al., 2011). Similar perspectives have also been adopted in the Americas (e.g. Brockman et al., 1992; Grossman et al., 2010; Kim et al., 2010). Research on microbial communities in soils was already used in investigations

S. Peters et al. / Quaternary International 324 (2014) 162e171

on Pleistocene and Holocene soils and sediments, as well as in studies to reconstruct environmental dynamics in a long-term perspective (Zvyagintsev et al., 1985; Colwell, 1989; Khlebnikova et al., 1990; Friedmann, 1993; Rivkina et al., 2004; Gilichinsky et al., 2005; Bai et al., 2006; Lacelle et al., 2011). Most of these studies were situated in permafrost soils of the tundra, Arctic, or Antarctic. Another study sampled drill cores through sediments to 100 m depth. The present case study is based on samples from a high-mountain site in the North Caucasus (Russia), where equally favorable conditions of a moist and cool environment hold out the prospect of good preservation of microbiological soil components.

Table 1 Physico-chemical properties of the cultural layer and background soils in the analyzed depth of the soil trenches. Soil samples

Corg % CaCO3 P2O5 mg/ pH (CaCl2) Particle-size 100 g soil distribution [%] 0.002 - < <0.002 mm 0.063 mm

e6 e7 e8 e9 e1 e3 e4 e5 e 13 e 11 e 12 e 10 e 14 e 17 (0e20 cm) ST e 17 (20e40 cm) ST e 17 (40e60 cm) ST e 17 (60e80 cm) ST ST ST ST ST ST ST ST ST ST ST ST ST ST

2.3 2.2 2.9 4.9 5.8 3.7 5.9 4.5 3.1 4.3 4.7 4.5 6.1 5.0

1.4 1.5 3.1 2.8 2.4 2.4 2.4 1.5 2.0 3.1 7.1 2.0 2.0 1.5

2.0 1.3 1.9 2.7 2.4 2.3 5.3 4.7 3.8 2.8 3.6 9.6 7.6 6.6

6.5 6.3 7.0 6.8 6.9 7.1 6.7 6.6 6.8 7.0 7.1 6.6 6.5 5.5

21 19 18 25 23 25 27 24 25 30 30 28 26 28

48 32 29 44 39 40 43 46 45 37 30 37 38 40

3.7

1.3

4.0

5.6

28

41

3.7

1.1

4.0

5.7

28

40

3.5

1.5

4.8

6.2

28

41

Table 2 Microbiological properties of the cultural layer and background soils in the analyzed depth of the soil trenches. Soil samples

e6 e7 e8 e9 e1 e3 e4 e5 e 13 e 11 e 12 e 10 e 14 e 17 (0e20 cm) ST e 17 (20e40 cm) ST e 17 (40e60 cm) ST e 17 (60e80 cm) ST ST ST ST ST ST ST ST ST ST ST ST ST ST

Vbas

Vsir

Cact

Cmic

Cact/Cmic

Cmik/Corg

mg C*g-1 soil *h-1

mg*g-1 soil

%

0.18 0.34 0.37 0.33 0.16 0.32 0.81 0.37 0.41 0.11 0.05 0.53 0.29 0.87

1.03 2.01 1.58 2.82 5.84 2.99 4.96 4.29 7.91 3.27 3.43 6.43 7.43 10.54

83.53 163.39 128.62 229.03 473.34 242.81 268.26 347.73 640.65 264.86 277.96 520.86 601.89 853.98

8356.47 5730.20 6200.17 3957.86 2330.66 3347.71 3177.26 3719.08 2886.77 3808.62 2520.82 3728.83 3097.61 6224.19

1.00 2.85 2.07 5.79 20.31 7.25 8.44 9.35 22.19 6.95 11.03 13.97 19.43 13.72

36.33 26.05 21.38 8.08 4.02 9.05 5.39 8.26 9.31 8.86 5.36 8.29 5.08 12.45

0.67

4.92

398.43

5785.60

6.89

15.64

0.52

1.75

141.72

5628.18

2.52

15.21

0.68

3.40

275.36

3671.43

7.50

10.49

163

Table 3 Microbiological parameters and organic carbon content of background soils in comparison to settlement soils and soils near settlement. Soil samples

Corg [%]

Cmic/Corg [%]

Cact/Cmic [%]

Reference soils Soil near settlement Settlement soils

2.4 4.9 4.5

27.2 8.1 7.1

1.8 5.8 11.5

During the last decade, the study of microbial communities in paleosols was transferred by Russian soil scientists to research on archaeological monuments. Especially, soils buried under burial mounds have been investigated (Demkina et al., 2000, 2004, 2007, 2010a, 2010b; Khomutova et al., 2004, 2011; Kashirskaya et al., 2009, 2010). Demkin (1997) has pointed out that paleosols retain some of their characteristics after being buried. They depend on the degree of conservation, which is, among other factors, reflected by the microbial characteristics (Demkina et al., 2007). Most of the cited studies focus on chronological and spatial variability of microbial soil characteristics, and discuss them within the framework of paleoclimate studies. As a dynamic integrated soil component, microbial communities are highly sensitive to changes of the environment. Concurrently, soil microorganisms manage to survive under adverse environmental conditions such as lack of nutrition or other unfavorable settings. They retreat into a dormant state and thus survive indefinitely long periods of time (Xu et al., 1982; Roszak and Colwell, 1987; Demkina et al., 2000, 2008; Khomutova et al., 2007). It is this factor which allows the use of microbiological methods in archaeopedological investigations. The characteristics of microbial soil communities do not only reflect the conditions of soil formation, i.e. the paleoecological perspective. They are also precise indicators of anthropogenic impact during soil formation in an ancient settlement. Based on the fact that microbial communities react specifically to the input of nutrients in the form of organic substances into soil, variations of quantitative and qualitative aspects can be expected depending on the intensity and mode of human activities in the past. It is most likely that the anthropogenic factor is the crucial one in soil formation within a settlement during its formation and use. It will therefore have a considerable impact on soil microbiological properties. When superimposed by layers of non-anthropogenic soil formation, we suggest that the characteristics of microbial communities in prehistoric cultural layers are preserved. They form a kind of archive both of environmental and anthropogenic conditions during the period of their formation. Their variation in layers of a similar age can be taken as a spatial indicator for activity areas, adding very precise statements about the nature of activities in the past. Bioindication as a new aspect in archaeopedology, however, opens a wide-ranging perspective to study human activities in the past. To date, mycological features have been shown as most promising sources of information (Marfenina et al., 2003). Fungi are highly sensitive to environmental variations, and in specific conditions form physiologically and structurally characteristic communities (Ivanova et al., 2006). Marfenina et al. (2001) presented a study of mycological characteristics of cultural layers from settlements. Spatial variation in the presence of keratin-decomposing fungi in sediments of streets, living-floors in and outside houses, or from wall-fillings revealed differences in the activities and the intensity of anthropogenic impact (Ivanova et al., 2006). These fungi utilize keratin, a very resistant protein in human and animal fibers (epidermis, hoof and horn material, fur, hair and feathers) as the sole source of C, N, S, and energy (Korni11owicz-Kowalska and Bohacz, 2011). Keratinolytic fungi intensively degrade native keratin, and are represented

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by dermatophytes pathogenic to humans and animals. The second nutrient-specialized group, keratinophilic fungi, utilizes only nonkeratinous components of keratin matter or the products of keratin (Ulfig, 2003; Korni11owicz-Kowalska and Bohacz, 2011). The term keratinophilicity is an ecological equivalent to microbiological keratinolycity (Korni11owicz-Kowalska and Bohacz, 2011). Several studies outside archaeology conduct analysis on isolation and classification of keratin-decomposing dermatophyts in soil (e.g. Vanbreuseghem, 1952; Ajello et al., 1965; Battelli et al., 1978; Kachuei et al., 2012; Bohacz and Korni11owicz-Kowalska, 2012). The keratinophilic fungi usually settle on dead fibers, which mix with the ground (Ivanova et al., 2006) and thus considerably change the quantity and quality of a microbial pool in the soil. Consequently, areas for increased presence of keratin-decomposing fungi in an archaeological context can be expected where animals have been present over a long period or in large quantities, such as stables, open corrals, or other places related to herding. Human hair, epidermis fibers, leather, cloth, and furs similarly are keratin fiber sources. Thus, living rooms should reveal higher keratindecomposing fungi concentrations. Secondary transport of fibers by wind or other disturbances must be considered as well. Another bio-indicator of ancient anthropogenic impact could be dark-colored soil fungi. Dark-colored fungi mycelia contains melanin pigments, which protect the cell from harmful hygric and thermic conditions, lack of nutritional elements (Bloomfield and Alexander, 1967; Butler and Day, 1998; Robinson, 2001), excessive sun exposure and industrial pollution (Zhdanova and Vasilevskaya, 1988), soil compaction, and other unfavorable effects (Marfenina et al., 1988; Mirchink, 1988; Terekhova et al., 1994). Their predominance in ancient palaeosols has been noted (Borisov et al., 2006; Demkina et al., 2010a,b). This paper presents a study of microbial characteristics of living floors of different contexts from an archaeological site and its surroundings. The site Kabardinka 2 in the North Caucasus (Russia) (Fig. 1) date to the Late Bronze Age (16th e 10th century BC) and belong to a community which specialized in intensive animal farming (Reinhold et al., 2007; Belinskiy et al., 2009). The soil studies on microbiological aspects are part of interdisciplinary research that has incorporated other non-destructive prospection methods such as geophysics (Fassbinder et al., 2007) and

excavation. Soil studies integrated physical and chemical aspects, microelement studies, and microbiological aspects, and are presented in this paper. It is one of the largest comprehensive studies on anthropogenic soils in prehistoric settlements to date, not only in Russia. 2. Objects and methods 2.1. Site setting, ecological and archaeological context The case study at Kabardinka 2 is part of a landscape archaeological research program concerned with development of human settlement in a high-mountain plateau starting in the 3rd millennium BC (Fig. 2). At the start of Late Bronze Age in the mid-2nd millennium BC, a part of former mobile herding communities became sedentary. In the 14th/13th century BC the small-scale settlement system was transferred into a dense system of settlements with large central places, around which a ring of houses was situated (Fig. 3). They are a specific adaptation to intensive herding. This project could identify for the first time a mountain agricultural economy, with shifting of animals in summer and stabling them inside the houses in winter. The phenomenon of Late Bronze Age settlements specialized on herding economy is limited to a high-mountain plateau about 60 km north of Mt. Elbrus massif and the main Caucasian range (Fig. 2). The sites are found at heights between 1400 and 2400 masl on flat slopes with northern exposure. Kabardinka 2 is one of the lowest sites, situated at an altitude of 1450 masl. The climate is moderate continental; mean temperature is 7.2
C, and mean precipitation is about 620 mm. During the 2nd millennium BC, temperature might have been slightly lower, as recorded for southeast Europe and the Caucasus (Kvavadze et al., 1997; Davis et al., 2003). The soil surrounding the sites is a mountain chernozem with a humus content of 5e8% (Egorov et al., 1977), today totally covered by mountain steppe vegetation. Test trenches outside the ruins revealed a soil depth of 30e50 cm, while in shallow depressions bedrock was found at 80 cm depth. The bedrock substrate is Lower Cretaceous calcareous sandstone (Andruschuk, 1968). The specific locations of soil sampling were related to a research strategy based on maximal non-destructive prospecting.

Fig. 1. Location of the investigated archaeological site in the Northern Caucasus (Russia).

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165

Fig. 2. Location of the site Kabardinka 2 and of archaeological phenomena on the high plateau of the Kabardian high-mountain chain.

Archaeological work continued from 2004 to 2008 (Fassbinder et al., 2007; Reinhold et al., 2007; Belinskiy et al., 2009). The ruins of the Late Bronze Age stone architecture were visible and documented in the topography. Most houses had two rooms each, with the entrances directed towards the outside of the settlement. Parallel to archaeological investigations, large-scale magnetometry and georadar surveys were carried out in order to locate structures and areas of supposed higher and lower anthropogenic impact. Two houses and a dump area were chosen for excavation, and three areas totalling 1500 m2 as well as 17 micro-trenches inside and outside houses have been sampled for soil studies. 2.2. Sampling strategies Soil samples at the site were taken in 2007 from the cultural layer, which is part of the modern soil and does not form a specific horizon, due to the limited thickness of overlapping sediments. When the site was abandoned in the 10th/9th century BC, soil formation processes started again. The area as surface horizon was exposed to denudation and accumulation. Today the cultural layer forms the lower part of soil profile (AC horizon of the modern Chernozem soil). It contains many cultural remains, such as ceramics, animal bones, and artifacts. Outside the excavation areas, the time span of the cultural layer is difficult to determine. In the excavated house 14, however, it was possible to differentiate two accumulation horizons dating between the 14th and 11th/10th

century BC, as well as two destruction layers from the 10th/9th century BC. We can assume that sampling positions at the lower part of the cultural layers thus reflect a period related to the lifespan of the house, while higher positions with abundant stone debris taphonomically relate to the destruction and decomposition period. To avoid destruction of archaeological features, sampling strategies focused on micro-trenches of 30  30 cm. These microtrenches were located both within different rooms of three visible houses and directly outside of them (Fig. 3). Similarly, a fourth house was sampled that had been located geophysically. This building is not part of the ring of houses around the central place and thus is an exceptional location. Two micro-trenches were located on the central place, and one at the dump area east of the settlement. The cultural layer is situated directly on bedrock and varies between 5 and 15 cm depth. Bulk samples were taken in all four sections of the micro-trenches from the basal 10 cm using sterile gloves and fumigated tools to avoid contamination. In micro-trench ST-17, systematic sampling each 20 cm was applied to assess the cultural layer as the major object of anthropogenic impact, i.e. aberration in the microbial community components and composition. To correlate results from cultural layers and unmodified soils, we systematically sampled soils outside the area of anthropogenic impact. Three more trenches located 70e200 m distant but in similar relief positions allowed characterization of the total

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Fig. 3. Disposition of Kabardinka 2 showing the location of soil trenches for sampling.

microbiological characteristics of untouched soil sequences. As in the settlement trenches, samples of the reference soils were taken from 10 cm above bedrock. 2.3. Methods 2.3.1. Physical and chemical analyses To measure pH, phosphorus, organic carbon and pedogenic carbonates content in soil samples we used standard laboratory techniques widely applied in Russia (Arinushkina, 1970). Soil texture was determined according to DIN ISO 11277 (2002), determination of particle size distribution in mineral soil material. 2.3.2. Microbiological analyses Microbiological analysis comprised the rate of basal soil respiration (Vbas), the substrate-induced respiration (Vsir), the biomass of active microorganisms (Cact), the total microbial biomass (Cmic) as well as the fungal mycelium biomass. The last step was a quantitative evaluation of keratinophilic fungi. Basal soil respiration (Vbas) following Anderson and Domsch (1978): After incubation for 6e8 h by 22
C of field-moist soil, the emitted CO2 was determined in a Chrom-5 GC with a katharometer

as a detector. The rate of basal respiration was calculated in mg Ce CO2 * g1 * h1. Substrate-induced respiration (Vsir) was measured similarly to Vbas, but soil samples were moistened using a glucose solution. Incubation lasted for 3e4 h (Ananyeva et al., 1993). Carbon content in active microbial biomass (Cact) (cells that respond to the added glucose) was calculated by the rate of substrate-induced respiration using the conversion factor 40.04 (Anderson and Domsch, 1978). The total microbial biomass (Cmic) was determined by luminescence microscopy using a fluorescent stain DAPI. Under a microscope (LUMAM I 2 LOMO), the cells were counted in 50 visual fields per glass (Kashirskaya, 2006). Each sample was analyzed in triplicate. The number of cells was calculated per gram of soil. The total biomass (mg C*g1) was then calculated assuming that the carbon content summed to 50% of the cell mass (Kashirskaya, 2006). On this basis, the ratio of active biomass to total biomass, as well as the ratio of total microbial carbon to total organic carbon, were calculated. Fungal mycelium biomass was determined by the membrane filter method (Aseeva et al., 1991) using a dyed AO VLADISART FMNC membrane filter. The hyphal length of the fungi was measured microscopically. For additional quantitative aspects of

S. Peters et al. / Quaternary International 324 (2014) 162e171

the microbial communities, the proportion between the non- and dark-colored mycelium and the sum of the total mycelia biomass within the total microbial biomass was calculated. Quantitative analysis of keratinophilic fungi. To analyse keratinophilic fungi, 25 g of soil in a sterile Petri dish was moistened to 70%. Keratin fibers in the form of 15 small pieces of hair were added. The dishes were sealed and stored under constant temperature of 22
C. In some cases, additional moistening was necessary. The analysis was performed three times, and hair and hyphal length were computed by quantitative correlation (Zvyagintsev et al., 1980). The activity of keratinophilic fungi was evaluated on a scale of 1e5. The lowest activity rate, 1, relates to single fungi colonies, while 5 represents total overgrowth with fungi colonies.

3. Results and discussion 3.1. Physical and chemical properties A comparative characterization of physical and chemical properties of samples from the cultural layer reveals several differences with the unmodified background soils from the surroundings (Table 1). Although pH-values of 5.5e7.1 are nearly similar within and outside the settlement, the cultural layer is characterized by higher rates of organic components and mobile phosphates. Maximal phosphate content revealed the dump area (ST-10) east of the architecture (Table 1). This suggests a high impact of organic, phosphorus-rich substances such as animal remains and bones. Elevated phosphate values caused by an increased input of organic plant materials can be excluded. This would correlate with a synchronic rise of organic carbon due to the accumulation of humus, which was not observed. Organic carbon contents here are similar to the reference soils. Rooms of the largest house (15), with three rooms and two attached chambers, have been sampled. In room 15/1 (ST-4) the phosphate content and organic carbon content increased sharply, while in the neighboring rooms 15 (ST-3) and 15/2 (ST-5) both parameters are markedly lower and rather similar to the reference soils outside (Table 1). The most likely interpretation is that room 15/1 was used as a room with intensive human activities, while the others were less or differently used. Interesting results were found in micro-trench ST-17, in a small room attached to house 15 (Fig. 3). This micro-trench was sampled in 20 cm steps and revealed variable chemical properties (Table 1). Due to its location in a depression and protected by the wall of house 15, the erosion rate was low. Thus, a greater quantity of silty material was accumulated. The upper 20 cm reveal high phosphate and organic carbon values similar to the modern background soils decreasing with depth, but phosphate increases in the lowest sample. This horizon is the cultural level of settlement, and the upper horizons have been formed by infill of eroded material. Here, the magnetic measurements (Fig. 3) display strong negative anomalies caused by intensive human impact. House 15 was built separate from the core area of the settlement. A clear cutting of former debris layers by its walls is apparent from the sharp variations of magnetic and non-magnetic features in the magnetometry analysis. Thus it is most likely that this house was added to the settlement after its first construction phase and stands on an area of dumping waste of the first settlers. The soil components of ST-17 thus should be compared to the test trenches at the dump area on the opposite side of the site. The trenches inside the house, however, can be related to its operational period. Contents of phosphate in the cultural layer of the non-visible house 26 (ST-1) that is situated apart from the other buildings are very low (Table 1). On the other hand, high organic carbon content

167

(Table 1) points towards an intensive input of plant material that led to humus accumulation. In the middle of the central place (ST-13) both phosphate and organic carbon are higher than in the reference soils, yet not extremely elevated (Table 1). This could be due to low input of organic substances both from animal or plant origin. Alternatively, such material may have been regularly removed (Reinhold et al., 2007). In a test trench on the central place, the excavation revealed a very thin cultural layer of only 2e5 cm, which supports the latter interpretation. In contrast, another sampling position at the northern end of the central place (ST-14) exposed significantly increased values of organic carbon and phosphate (Table 1). These soils have been considerably affected by human activities and perhaps very specific practices. In house 7, both rooms reveal high organic carbon contents in the cultural layers, although in the outer room oriented to the surroundings (ST-12), phosphate levels are comparatively high (Table 1). As in house 15, the differences point towards dissimilar activities in both rooms. 3.2. Microbiological characteristics of the studied soils 3.2.1. Basal and substrate-induced respiration A basic characteristic to analyze soil microbial communities is the parameter of respiration activity. Basal respiration (Vbas) does not show any differences between reference and settlement soils (Table 2). Likewise, no variation can be found in the samples from the settlement, due to identical conditions while taking samples and a similar depth of sampling. Thus, all samples reflect the modern bioclimatic conditions of the sampling period in October 2007. In the vertical samples of micro-trench ST-17, the maximum rate of basal respiration (0.87 mg C*g1) was detected in the surface horizon (Table 2). The rate decreases with depth, but in house 15/1 (ST-4) with high organic carbon and phosphate values (Table 1), the basal respiration rate (0.81 mg C*g1) is as high as in the surface horizon (Table 2). In contrast to the basal respiration rate, substrate-induced respiration (Vsir) differs between samples from the same depth taken inside and outside the settlement. Vsir characterizes the biomass of microorganisms which are in the active state and which can reflect additional glucose. Values of 1.03e2.01 mg C*g1 * h1 in reference soils compare to rates of 2.99e7.91 mg C*g1 * h1 from intra-site samples. Moreover, reference samples near the settlement such as from micro-trench ST-9 (Fig. 3) have higher rates (2.82 mg C * g1) than those that are more distant (Table 2). The vertical measurement in micro-trench ST-17 reveals a decrease of Vsir-rates with depth from 10.54 mg C * g1e1.75 mg C * g1, with a sharp increase (3.40 mg C * g1) in the buried cultural layer (Table 2). In general, the highest Vsir-rates have been found in both trenches from the central place ST-13, ST-14 (7.91 and 7.43 mg C * g1 respectively), the dump area (ST-10) (6.43 mg C * g1), and inside the separate house 26 (ST-1) (5.84 mg C * g1) (Table 2). Comparing basal and substrate-induced respiration, all horizons seems to generally reflect an insufficient supply of easily available organic material for microorganisms. When glucose was added, respiration rates increased fifteen-fold. In the soils from house 7 (ST-11/12) and 26 (ST-1), however, multiplication rates of 36/31 and 65 respectively were noticed. In these cases, a much higher quantity of microbe cells could be reactivated than at other points of the settlement or surroundings. Although carbon content at these locations varies, all phosphate values are rather low. These facts reflect a much higher input of persistent organic compounds, e.g. lignin or cellulose, than that of organic residues from animals, such as fibers or bones. In the reference soils as well as in the rooms of house 15, differences between Vbas and Vsir are low. Glucose has a

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S. Peters et al. / Quaternary International 324 (2014) 162e171

multiplication rate of about 6e9, which points towards another source of organic material added to the ground in prehistory. Thus, the substrate-induced respiration generally seems to reflect the former supply of organic material for microorganisms and may be used to indicate former human activity. 3.2.2. Active and total microbial biomass Correspondingly to the substrate-induced respiration rates, the biomass of actively metabolic cells is highest at locations ST-13, ST14, ST-10, ST-1 (640.65, 601.89, 520.86 and 473.34 mg C * g1 respectively) (Table 2). In general, the active biomass of background soils is 2e3 times lower than within the settlement. Considering the ratio of active cells to the total microbial biomass, on the other hand, a much higher quantity of total biomass is found in the reference soils (3957.86e8356.47 mg C * g1) (Table 2). In anthropogenically stressed soils of the settlement, the entire microbial biomass is significantly lower (2330.66e3808.62 mg C * g1) than in the reference soil outside, but the proportion of active biomass (Cact/Cmic) is much higher (Table 3). Most cells (97%) of the microbial communities in the anthropogenically unmodified soils remain in inactive forms. The highest percentage of active biomass to the total microbial biomass were found at the central place (ST-13 (22.2%)), in house 26 (ST-1 (20.3%)) and the northern end of the central place (ST-14 (19.4%)). The outer room of house 7 (ST-12) and the dump area (ST10) show high shares of active cells as well (11 and 14% respectively). In micro-trench 17 a similar gradient shows a decrease of percentage with the depth and a rise in the lowest horizon (Table 2). The level of active metabolic cells at the recent surface is 14%, comparable to the proportion in the cultural layers at the dump area (ST-10) and in house 7 (ST-12). 3.2.3. Microbial carbon Cmic and total organic carbon Corg Cultural layers and unmodified background soils revealed significant variations of active biomass with higher values in the first one (Table 3). The percentage of total microbial carbon Cmic of the total organic carbon Corg is higher in the reference soils than in the site (Table 3). The Corg values increases towards the settlement, while the share of the microbial carbon decreases (Table 3) and changes in the composition occur, as more cells can be reactivated. The microbial communities change, not only quantitatively but also qualitatively under anthropogenic influence.

3.2.4. Mycological aspects Among the most important aspects detecting alterations in the microbial communities of soils are the characteristics of soil fungi. This study intend to differentiate more precisely the modalities generally subsumed under the term “human impact” using the mycological qualities of the microbial-pool from soils at Kabardinka 2. Based on the fact that environmental conditions (topography, soil, vegetation, humidity, temperature, current land use, etc.) for fungal growth in different parts of the settlement and beyond are in general the same, variations in the biomass of fungal mycelium, percentage of dark-colored mycelium and the number of keratindegrading fungi indicate anthropogenic imprint on the soils in the past. Following the preliminary studies of Marfenina et al. (2001, 2008) which used the keratinophilic fungi as principal markers for animal presence, variations should point to those areas where animals had been present at some time during the formation of the cultural layer. In most reference soils, fungal mycelium biomass is comparatively low and ranges from 82.4 to 235.2 mg C/g soil (Fig. 4). The percentage of dark-pigmented hyphae in these soils is about 70e 83%. Only in the reference micro-trench ST-8 200 m west of the site is fungal mycelium biomass (314 mg/g soil) comparatively high. Here, dark-pigmented hyphae comprise 202.9 mg/g soil or 65% of the total sum (Fig. 4). With regard to the total fungal mycelium biomass, cultural layers in house 15/1 (ST-4) and both rooms in house 7 (ST-11/12) can be compared to the reference background soils (240.8; 230.6, and 146.3 mg/g soil, respectively) (Fig. 4). This means that these locations had conditions which did not enable rapid growth of soil fungi. The percentage of dark-pigmented hyphae in sample ST-4 (house 15/1) and the inner room of house 7 (ST-11) differ significantly from the total fungal mycelium biomass (Fig. 4), at 43 and 59%, respectively. In these rooms, keratinophilic fungi are as scarce as in the reference soils (Fig. 5). In contrast, in the outer room of house 7 (ST-12), 100% of the fungal mycelium biomass consists of dark-pigmented hyphae (Fig. 4). Here, the presence of keratinophilic fungi is clearly higher than in the neighboring room (Fig. 5). Different activities related to both rooms can be assumed. According to Marfenina et al. (2008), the keratinophilic fungi in any case point towards the presence of animals in or directly outside the room as sources for keratin fibers.

µg C/g dry soil

a

b

600 550 500 450 400 350 300 250 200 150 100 50 0 1

2

3

4

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Fig. 4. The content and structure of fungal mycelium in the cultural layer of archaeological site Kabardinka 2 and their analogues beyond the settlement (a e biomass of dark fungal mycelium; b e total biomass of fungal mycelium; 1 e reference soil ST-6; 2 e reference soil ST-7; 3 e reference soil ST-8; 4 e soil near settlement ST-9; 5 e house 26 ST-1; 6 e house 15 ST-3; 7 e house 15/1 St-4; 8 e house 15/2 St-5; 9 e central place ST-13; 10 e house 7 ST-11; 11 e house 7a ST-12; 12 e dump area ST-10; 13 e northern part of central place ST14).

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The fungal mycelium biomass in the two other rooms of house 15 (ST-3/5), as well as at both investigated spots on the dump area (ST-10) and the northern part of the central place (ST-14), have values of 388.3, 305.9, 276.2, and 335.6 mg/g soil respectively. At first sight, these places offer ecological conditions where fungi can thrive equally. However, the ratio of dark-pigmented hyphae in the rooms of house 15 is 65% and 57%, while at the dump area and the northern part of the central place almost the total sum of fungi is dark-pigmented mycelium (93 and 86%). A difference in the activities that lead to varying of nutrition supply at these areas in the site can be supposed, which is also confirmed by keratinophilic fungi (Fig. 5). Very low activity of these fungi was found in the two rooms of house 15, which can be read as the operational part of the house. The locations had shown no significant indicators for human activities. In the northern part of the central place, on the other hand, keratinophilic fungi activity is as high as in the outer room of house 7 (Fig. 5). In 2005, a preliminarily trial trench on the central place had, for the first time, revealed the general presence of these kind of fungi at the site (Reinhold et al., 2007). The systematically analyzed sample from 2007 in micro-trench ST-13 revealed a total mycelium biomass of 468.3 mg/g soil. It consists nearly completely of dark, melanin-containing mycelium (95%). Increase of melanincontaining mycelium is a result of suppression of soil fungi in consequence of the extremely strong human impact on soil at ancient settlements. Coth the high presence of keratinophilic fungi and shallow soil depth at the central place may be explained by the presence of large quantities of livestock. The particles of their horns, hooves, and hair fibers directly fall into the soil and serve as food for keratinophilic fungi. The removal of organic material could thus be related with producing dung-bricks for heating, as is still practiced in Caucasia and Eastern Anatolia. The highest keratinophilic fungi concentration at Kabardinka was found at the dump area (ST-10) (Fig. 5). As the later excavations showed by the presence of dump layers, together with much other organic waste, large quantities of keratin substances from animal or human origin had entered the soil here. Unexpectedly high values of total fungal mycelium biomass were detected in house 26 (ST-1) (526.3 mg/g soil). However, the ratio of dark-pigmented mycelium is 63% of the total sum and is thus significantly lower (Fig. 4). The activity of keratinophilic fungi is not elevated in this house. In addition to both the high organic carbon and low phosphate values, and the low microbial biomass, the low amount of keratinophilic fungi indicate activities that did not include the stabling of animals e.g. slaughtering or butchering, but rather are related to the input of plant organic substances into the ground.

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Near house 15, the samples in micro-trench ST-17 revealed the highest values of fungal mycelium biomass at the modern surface (334.0 mg C/g soil) (Fig. 6). Here, keratinophilic fungi are present, which must be related to grazing of modern herds at the site. The ratios both of fungi and of keratinophilic species, however, decrease with depth (Fig. 5; Fig 6). In the lowest samples at 40e80 cm depth, the values increase (241.3e211. 7 mg C/g soil). The percentage of dark-colored mycelium is slightly less in the upper layer and increases with depth (Fig. 6). In all lower samples, the total sum of fungi consists almost entirely of dark-colored hyphens (Fig. 6). Thus the elevated level of dark-colored fungi may serve as indicator of a cultural layer in cases where no morphological features are revealed in the soil profile. The vertical section indicates that the analyzed horizons in the samples reflect the ancient anthropogenic influence, i.e. a prehistoric cultural imprint, and not a recent impact by modern herding activities in the region. The analysis clearly reveals that the disturbances of soil microbiological composition due to human impact approximately 3000 years ago still affects the soil characteristics. Quantities of microorganisms and their state visibly differ from neighbouring, untouched areas. In soils buried below burial mounds, such diversities can be explained by paleoclimate changes (Khomutova et al., 2004; Demkina et al., 2008). This differs in the buried anthropogenicallyinfluenced palaeosols. Climatic change since the Bronze Age cannot be held responsible for the diversification of the soil microorganisms. Similar climatic conditions affected both soils within and outside the settlement. The cultural layer was not buried below an artificial construction and thus was subject to the same bioclimatic dynamics as the surroundings. The variations must be understood as a result of human impact in the past. Artificially adding organic matter of different origin e animal, human or plant waste e offered the ancient microorganisms a variety of additional sources of nutrition, including easilyaccessible substances. Natural sustenance conditions of the microorganisms in comparison with untouched areas thus were considerably altered. When the settlement had been abandoned, the additional nutrition influx of organic matter stopped. This lead to shortage of nourishment, to which the microorganisms had to adapt by a reaction, such as transfer into a dormant state. When extra nourishment such as glucose was added artificially, more cells could be revived in the settlement soils than in the soils from outside. In the reference soils where no additional organic matter had been added, fewer cells reacted to the addition of glucose. Obviously, they use different sources of nutrition. The general high content of active microbial biomass as well as low level of total microbial biomass in the cultural layers is most likely a result of the presence of a higher percentage of inactive, deceased or mummified cells in the background soils. Khomutova

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et al. (2011) noticed this effect in palaeosols buried below burial mounds in the Privolzhkaja plain (Lower Volga region). Due to the positive nutrition conditions in the cultural layers, the mycelium biomass is equally higher than in the reference soils of the surroundings. However, several areas of the site are characterised by dark pigmented fungi hyphae, up to a percentage of 100%. It is known that dark colored fungi in large quantities are observed in urban soils (Kul’ko and Marfenina, 2001), buried soils of steppe kurgans (Borisov et al., 2006; Demkina et al., 2010b), but also in mountains with high insolation (Mirchink, 1988). The melanin containing fungi can withstand unfavourable environmental factors, including the lack of carbon (Borisov et al., 2006; Demkina et al., 2010a). This argument, however, cannot hold for the soils at Kabardinka 2, as their organic carbon content is much higher than that of the surrounding soils. Much more likely, this is related to the anthropogenic stress that these soils have been exposed to in the past. This is more apparent in not all areas in the sites revealed similar alterations. Thus, not all areas in the site were exposed to the same anthropogenic pressure. On the central place, the northern part of central place, the dump area and in the outer room of house 7, both the percentages of dark pigmented hyphae from the total mycelial biomass and quantities of keratinophilic fungi increased. Their presence indicates an enlarged influx of keratin fibres into the soil, however, without a precise relationship to any specific origin. Such fibres can come from animal presence, but human substances, shifted materials, woollen carpets or felt similarly might have led to the increased level of keratin. In any case, recognizing keratinophilic fungi in houses or other areas of a settlement opens up a wide spectrum of information, even without a more precise classification of the species as found in the work of Marfenina et al. (2003) and Ivanova et al. (2006). Focus on soil analytical methods such as urea bacterial activities (Borisov et al., in press) allow more precise location of human activities. 4. Summary At the Bronze Age settlement of Kabardinka 2 in the North Caucasus (Russia), for the first time systematic and large-scale microbiological soil analysis has been introduced to interdisciplinary archaeological research. This methodological approach permits a characterisation of microbiological conditions in cultural layers and untouched natural soils. Nearly all investigated parameters reveal clear differences between settlement soils and soils which never had been exposed to anthropogenic impact. In the anthropogenic palaeosols, the organic carbon and phosphate content as well as the actively metabolic cells increased. Similarly, a higher mycelium biomass and percentage of kerationophilic fungi was found with simultaneously low rates of total microbial biomass. The microbiological parameters clearly reflect intensity and conduct of the human activities at the site in the past. Microorganisms, due to a particularly sensitive reaction to anthropogenic modification of natural living conditions, reveal a great potential for archaeological investigation. They sharply reflect the intensity of ancient activities and can link the range of interpretative hypotheses to practical aspects of ancient village life. Inside a settlement, the microbiological parameters vary according to soil composition and former organic matter. In consequence, heavily used areas such as the dump zone, living quarters or rooms, storage rooms, as well as the presence of large quantities of animals in the site was demonstrated. In the multidisciplinary perspective of the Kabardinka 2 excavation, microbiological soil analysis has proven to be an excellent method to detect and evaluate ancient activity areas. In combination with magnetometry, excavation and other classical as

well as microbiological investigations, this method has an extremely high heuristic value for the understanding of ancient ways of life.

Acknowledgments This work is part of a joint German-Russian research project at the Eurasia-Department, German Archaeological Institute in Berlin, Germany (PD Dr. Sabine Reinhold) in co-operation with the Institutes of Archaeology RAS, Moscow, Russia (Dr. Dmitri S. Korobov), the local department of antiquities GUP ‘Nasledie‘, Stavropol‘, Russia (Dr. Andrej B. Belinskiy) and the Institute of physicochemical and biological problems in soil science of the Russian Academy of Sciences, Puschchino (Dr. Alexander V. Borisov). It was financed by the German Research Foundation (N
RE 2688/1-3) and the Russian Foundation for Humanities (N
06-0192012a). For administrative and financial support we thank Dr. Anderj B. Belinskij, Stavropol’. Statistical and experimental support was contributed by PD Dr. Natal’ya N. Kashirskaya, Pushino. We are also very grateful to the referees for their important suggestions and comments.

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