Seasonal dynamics of soil microbial biomass in fragmented patches of subtropical humid forest of Jaintia hills in Meghalaya, Northeast India

Aim of the study: The aim of the study was to assess the seasonal dynamics of microbial biomass and its contribution to soil system along a fragment size gradient in subtropical humid forest of Meghalaya. Area of study: The study was conducted in forest fragments located at Jarain and adjoining areas in Jaintia Hills of Meghalaya, northeast India. Material and Methods: Forest fragments of sizes ranging from 3.8 to 105 ha were selected for the study and grouped into Small (< 5 ha), Medium (> 5 and < 15 ha), Large (>15 and < 50 ha) and Very Large (105 ha) classes. Three experimental plots each of 20 x 20 m were established at the forest edge and at 50 m distance assigned as ‘interior’ microsite in each of the fragments for sampling of soil. Soil samples (0-10 cm depth) from each of the experimental plots were collected in replicates on seasonal interval and microbial biomass was estimated by the fumigation extraction method. Important findings: Microbial biomass -C, -N and -P varied significantly (p< 0.05) between the fragment sizes, microsites and seasons. The microbial biomass was higher in the interior as compared to the edge. It was also high during the winter season. Overall, soil microbial biomass -C, -N and -P ranged from 260 to 969; 25 to 95 and 8 to 67 µg g -1 respectively. The contribution of microbial biomass -C, -N and -P to soil organic carbon, total Kjeldahl nitrogen and phosphorus ranged from 1.48 to 1.81 %, 2.54 to 4.54 % and 3.41 to 5.22 % respectively. Fragmentation alters the microenvironmental conditions and soil properties that in turn affect the microbial biomass. Highlights: This interaction of plant, soil and microbial community would gradually degrade in the fragments due to change in vegetation composition and structure, microclimatic conditions and soil physical and chemical properties. Our results suggests that microbial mediated ecosystem processes such as nutrient cycling are more susceptible to variation at the edge which may become unstable and unpredictable in forest fragments exposed to various human disturbances.


Introduction
Fragmentation of forests has been identified as a major cause of biodiversity loss.It leads to edge creation and alter the microclimatic conditions that may have deleterious effect on ecosystem structure and function (Chen et al., 1999;Ibanez et al., 2014).Forest edge experiences increased soil temperature, high evapotranspiration and reduced soil moisture content due to greater insolation (Camargo & Kapos, 1995).Edges are also exposed to high wind turbulence.Consequently, trees on the edge are prone to high mortality owing to physical damage as well as physiological stress (Laurance et al., 1999;2000).Thus forest fragmentation leads to a shift in ecological traits of dominant species and reduces the buffering capacity of microclimate at the edge (Zhu et al., 2004).The elevated rates of tree mortality and recruitment in fragmented forests drive the biomass (Laurance et al., 2014) and the litter quality that may alter carbon and nutrient return to the soil (Laurance et al., 1997;1998).Studies have shown that the litterfall increases in the forest edge with high nutrient concentration (Sizer et al., 2000) but often displays decrease decomposition rate (Moreno et al., 2014;Ruitta et al., 2012).Such changes may affect the soil nutrient balance in the edge (Bierregaard et al., 1992).As a result synergistic impact of shift in dominant plant community and microclimatic conditions directly or indirectly influen ce the resource availability on the soil (Haddad et al., 2015).Further, in fragmented forests, changes in microbial population and invertebrate activity may lead to reduced decomposition rates and may affect nutrient cycling (Yeong et al., 2016).
Soil microbial biomass plays an important role in the conversion of organically bound plant nutrient into inorganic forms (Singh et al., 1989).The role of microbial activities in regulating the nutrient dynamics is crucial in sustaining forest ecosystems.Soil microbial biomass is regulated by soil physical and chemical properties and microclimatic conditions (Chen et al., 2005;Devi & Yadava, 2006).Fragmentation and habitat degradation may change plant-soil-microbe interaction, influence soil nutrient dynamics and control the above ground community of the fragments (Lazaro-Nogal et al., 2012).Thus microclimatic conditions at the edge can further alter microbial dynamics and their role in biogeochemical cycles.As drier conditions will favour fungal-dominated soil microbial communities, leading to a deceleration of processes associated with the plant-soil-microbial system (Flores-Renteria et al., 2015).However, there are very few studies related to the effect of fragmentation on ecosystem functions particularly nutrient dynamics and microbial activity.Soil microbial biomass and activity and the faunal group (especially the nematodes) are often used as impor tant indicators to assess the ecosystem quality and health status after disturbances (Schloter et al., 2003).
The subtropical broadleaved humid forests of Meghalaya in northeast India distributed between 1000 m and 1800 m are highly fragmented due to anthropogenic activities such as mining, shifting cultivation, construction of roads and urbanization (Pao & Upadhaya, 2017).These forests in the state are currently characterized by small remnant patches scattered on strolling grasslands and areas that are usually inaccessible.Fragmentation changes the vegetation composition, community structure and microclimatic conditions and thus could be one of the important factors affecting the soil nutrient status and the microbial biomass (C, N and P).Although there are a number of studies that have established that climatic factors, soil pH and temperature influence mi crobial biomass in different forest types (Maithani et al., 1996;Singh & Kashyap, 2006;Singh et al., 2010), but the effect of fragmentation is poorly understood.We hypothesize that along a fragment size gradient soil microbial biomass would vary and hence affect nutrient addition to the soil with low biomass and nutrient in the small as compared to larger fragments.Therefore, the specific objectives of the present study was to assess the seasonal dynamics of soil microbial biomass and its contribution to soil organic matter and nutrient along a fragment size gradient in subtropical forests of Jaintia hills in Meghalaya, northeast India.

Study area
The study was conducted in Jarain (latitude 25°19' 20.45"N-25°19'25.55"N and longitude 92°07' 42. 33"E-92°09'23.09"E,altitudinal range 1000 -1100 m) in Jaintia Hills of Meghalaya, northeast India.The forest in the study area occurs in the form of pat ches and was once part of the same continuous forests that got fragmented over time due to human disturbances such as road construction, shifting cultivation and mining (Pao & Upadhaya, 2017).For the present study a total of 10 fragments were selected.The size of the fragments ranged from 3.79 to 105 ha (Table 1).All the selected sites were within a 6 km radius of Jarain village and had similar vegetation and soil.
The forest fragments were classified into four size classes viz.Small (< 5 ha), Medium (> 5 and < 15 ha), Large (> 15 and < 50 ha) and Very Large (105 ha), so that they represent a size gradient and were abbreviated as S, M, L and VL respectively.A set of three forest fragments were maintained for each fragment class (Table 1).However, in the case of the Very Large (VL), three replicated plots were maintained from a single large fragment.This was due to lack of large continuous tract of forests in the study area.
The vegetation of the area falls under subtropical broadleaved humid forest type (Champion & Seth, 1968).The vegetation is dense with a canopy height where trees rarely exceeds 18 m.(Pao et al., 2016).The ground vegetation is dominated by ferns, mosses, and species belonging to Balsaminaceae, Orchidaceae, Rubiaceae and Asteraceae.The vegetation characteristics of the studied fragments are shown in Table 1.
The climate of the area is tropical monsoonal and the rainfall is influenced both by the south-west and north-east winds.Four seasons -autumn (September -November), winter (December -February), spring (March -May) and rainy (June -August) are distinguishable.The mean annual rainfall during the period (January 2013 -December 2015) was 5550 mm.The a ve rage maximum and minimum temperature was 22 °C and 14 °C respectively (Figure 1).
The Jaintia Hills is a continuation of the Meghalaya Plateau formed from the remnants of Pre-cambrian Indian peninsular shield.The rocks are mostly of Precambrian origin with gneissic composition such as granites, schist, amphibolits and calc-silicate.The topography of the study area is more or less flat with rolling mounds representing plateau (Central Groundwater Board, 2013).The soil texture in all the studied fragments was loamy sand.Soil bulk density ranged from 0.98 to 1.10 g cm -3 whereas, the water holding capacity ranged from 50 to 59 % (Table 2).

Micro-environmental variables
The micro-environmental variables in the selected fragments at the edge and interior microsites were studied for a period of two years (October 2013 to July 2015).Light intensity, relative humidity, air-and soiltemperature were measured during October, January, April and July.These months correspond to autumn, winter, spring and rainy seasons, respectively.The measurements were taken between 9 and 10 a.m. for three consecutive days in each of the four seasons of sam pling.All the variables were measured randomly at three places in different microsites (edge and interior) in the selected fragments.Light intensity was measured by using Digital Luxmeter (TES 1332A), atmospheric temperature and relative humidity by Thermo-  hygrometer TH-103 (Mex-therm).Soil temperature was measured by digital soil thermometer (Multi-Thermometer) by inserting the probe into the soil to a depth of 10 cm.

Soil sampling
Three experimental plots each of 20 x 20 m were established at the forest edge (measured as the distance from the last tree at the edge to 20 m into the forest) and at 50 m distance assigned as 'interior' microsite in each of the fragments for sampling of soil.The soils were sampled to a depth of 10 cm after removal of litter layer.Three soil samples from each of the experimental plots were collected using an iron soil corer (6.5 cm diameter and 20 cm height) on seasonal basis from each fragment.Thus a total of nine samples each were retrieved from edge and interior microsites separately on every sampling period for each fragment sites.These samples were bulked together microsite wise to get a composite soil sample for each fragment sites.The samples were further divided into sub samples for analysis.The first part was used for determination of soil moisture content (SMC), pH, microbial biomass, ammonium-N (NH 4 + -N) and nitrate-N (NO 3 --N).The second part was air dried and grounded for determination of soil organic carbon (SOC), total Kjeldahl nitrogen (TKN) and extractable phosphorus (Ex.P).The data collected from both microsites and each fragment sites were pooled together and presented fragment class wise.

Laboratory analysis
SMC was determined gravimetrically by drying 10 g of fresh soil in a hot-air oven at 105 °C for 24 h and the result was expressed on oven-dry weight basis (Allen et al., 1974).Soil pH was determined by a digital pH meter taking 1: 2.5 suspension of soil and distilled water.NH 4 + -N was determined by the indophenol blue method after extracting the fresh soil samples with 1 N KCl solution (Allen et al., 1974).NO 3 --N was determined by the phenol disulfonic acid method from fresh soil samples extracted with deionized water (Allen et al., 1974).SOC was determined by colorimetric method following Anderson and Ingram (1993).TKN was determined by the semimicro Kjeldahl digestion method by digesting air dried sample with concentrated H 2 SO 4 using Kjeltabs as catalyst followed by distillation and titration (Allen et al., 1974).Extractable P (Ex.P) was determined by Molybdenum blue method after extracting the air dried soil with 0.5 M NaHCO 3 (Allen et al., 1974).
Soil microbial biomass -C, -N and -P was determined by the fumigation-extraction method.Three subsamples (10 g each) were fumigated by saturating with 10 mL of alcohol free liquid chloroform for lysing of cell while other three were unfumigated.The fumigated and unfumigated soils were extracted with 0.5 M K 2 SO 4 and filtered through Whatman filter pa per no.42.These filtrates were used for the determination of microbial biomass -C and -N.The organic carbon content in the extracts was determined by digesting the extract in H 2 SO 4 and 0.00667 M K 2 Cr 2 O 7 .The diges ted sample was titrated with acidified ammonium ferrous sulphate solution using an indicator (O-phenolphthaline monohydrate and ferrous sulphate hexahydrate).The volume of titrant consumed was deduced for estimation of organic carbon extracted (Vance et al., 1987).
Similarly, for microbial biomass nitrogen (MBN) the extracts were further digested with 4.4 mL of digestion mixture of selenium powder, lithium sulphate, hydrogen peroxide and H 2 SO 4 .The digested solu tion was extracted with distilled water in What man filter paper no.1.The filtrate was used to determine the ammonium released through distillation with 40 % NaOH.The distillate was collected in boric acid indicator till the permanent green colour develops.It was titrated against N/140 HCl till it turns into pink colour.Microbial biomass phosphorus (MBP) was determined by chloroform fumigation-extraction method (Brookes et al., 1982) and further extracted by ammonium molybdate blue method (Allen et al., 1974).Microbial biomass -C, -N and -P were calculated as: MBC (µg g -1 soil) = 2.64* Ec Where, Ec = difference between the amount of organic carbon extracted from the fumigated and nonfumigated soils expressed in µg C per g of soil 2.64 = the relationship between biomass C as measured by incubation method and amount of C extracted by 0.5 M K 2 SO 4 after chloroform treatment. MBN Where, a = amount of phosphorus (µg g -1 ) extracted from the unfumigated soil b = amount of inorganic phosphorus (µg g -1 ) extracted from the fumigated soil 0.40 = the fraction of biomass phosphorus mineralized and extracted in 0.5 M NaHCO 3 (Brookes et al., 1985).

Statistical analysis
The data on microclimatic variables (light intensity, relative humidity, air-and soil-temperature) and soil moisture content, pH, SOC, TKN and Ex.P, NH 4 + -N, NO 3 --N and microbial biomass C, N and P were subjected to two-way analysis of variance (ANOVA) of repeated measures.The analysis considered three factors: two factors included fragment size and microsites (between subjects) and one time factor included season (within subject) with four levels (autumn, winter, spring and rainy) spread across two years.Assumptions of ANOVA were met through test for normality of variables (Kolmogorov-Smirnov test), and homogeneity of group variances (Levene's test).Pearson's correlation was computed to understand the influence of fragment size, microclimatic conditions and soil properties on microbial biomass.All statistical analysis were performed using the software SPSS, version 13.

Micro-environmental variables and soil properties
Light intensity varied significantly (P< 0.05) across the seasons with higher values during spring and low in rainy season (Table 3 and 4).It was high in small patches and the edge as compared to larger patches and interior microsites (P< 0.05).The air-and soiltemperature showed a similar trend with higher values during spring and rainy and the lowest values were recorded during winter season.However, they did not vary significantly (P> 0.05) across the studied fragments but varied between microsites (P< 0.05).The air temperature ranged from 23 to 25 ºC while soil temperature ranged from 19 to 21ºC in the studied fragments.The soil moisture content showed a strong seasonality (P< 0.05) with higher values during rainy and lower during winter.It was higher in the larger patches (P< 0.05) and interior microsites as compared to small patches and edge microsites.It ranged from 19 to 29 % in the studied fragments.The soil in the studied fragments was acidic (pH 5.4 -5.7) and showed a marked seasonality (Table 3 and 4).It was more acidic (4.53 -4.88) during the rainy season.
The SOC showed a marked variation (P< 0.05) among the studied fragments, microsites and seasons (Table 3 and 4).It ranged from 144 to 613 g Kg -1 among the fragments.SOC content was higher in the interior microsites as compared to the edges in all the forest patches.TKN ranged from 7 to 48 g Kg -1 .Similarly, the values of Ex.P ranged from 6.54 to 8.36 µg g -1 .It varied significantly between the fragments and sea sons (P< 0.05).NO 3 --N was the dominant form of nitrogen in the study area.NH 4 + -N and NO 3 --N concentration ranged from 9.73 to 11.18 µg g -1 and 10 to 13 µg g -1 respectively (Table 3).The values were significantly (P< 0.05) higher in the interior -as compared to the edge -microsites (Table 4).

Soil microbial biomass
The MBC concentration varied significantly (P< 0.05) between the fragment size, microsites and seasons (Table 5).The values of MBC in the fragments ranged from 260 to 969 µg g -1 (Figure 2a).The range of values were 260 -748 µg g -1 in Small, 289 -781 µg g -1 in Medium, 362 -793 µg g -1 in Large and 351 -969 µg g -1 in Very Large fragments.The values were significantly higher (P< 0.05) in the forest interiors as compared to the edges (Table 5).MBC showed a marked seasonality (P< 0.05) with highest values during the winter season that gradually declined through the spring to its minimum level in the rainy season in all the fragment classes (Figure 2a).
The MBN varied significantly (P< 0.05) between the fragments classes and ranged from 28 to 92 µg g -1 (Figure 2b).It was significantly higher (P< 0.05) in the forest interior as compared to the edge (Table 5).The mean values in the edge along the fragment size gradient (from Small to Very Large) were 43, 44, 46 and 50 µg g -1 respectively.Similarly, the mean in the interior of the fragments were 51 (Small), 55 (Medium and Large) and 58 µg g -1 (Very Large).It showed a marked seasonality with low values during rainy season and high during winter (Figure 2b) in all the fragment classes.
Similarly, the microbial biomass phosphorus (MBP) values in the studied fragments ranged from 10 to 51µg g -1 .It varied significantly (P< 0.05) between the fragments classes.The average MBP values for edge and interior was 26 and 29 in Small, 30 and 33 in Medium, 29 and 32 in Large and 35 µg g -1 in Very Lar ge respectively.Although MBP increased significant ly with the increase in fragment size class, there was no significant difference between the microsites (P> 0.05) (Table 5).It showed a marked seasonality (P< 0.01) with low values during rainy season and high during winter (Figure 2c).

Microbial C/N and C/P
The microbial C/N ratio varied in the studied fragments.The value was high (11.92) at the edge of Large fragment class and low (10.29) in the interior  Small,11.79 and 10.69 in Medium,11.92 and 11.39 in Large and 11.55 and 11.04 in Very Large (Table 6).
Similarly, the microbial C/P ratio was high in the Larger fragment class in both the edge and interior (19) microsites.However, microbial C/P ratios did not show any consistent variation between the two microsites among the fragment classes except Very large (Table 6).

Contribution of microbial biomass to soil carbon, nitrogen and phosphorous
The soil microbial biomass -C, -N and -P as proportions to SOC, TKN and Ex.P in the studied fragments is shown in Table 6.The percentage contribution of MBC to SOC was 1.48, 1.62, 1.58 and 1.62 for the edge microsites of Small, Medium, Lar ge and Very Large fragments respectively, while the cor res ponding values for the interior microsites were 1.54, 1.81, 1.70 and 1.66.Generally, the contribution of MBC to SOC was higher in the   The F values marked with * are significant at P< 0.05, ns = not significant.
Table 5. Two-way analysis of variance (ANOVA) showing the effects of fragment size class, microsites and seasons on soil microbial biomass -C, -N and -P.The F values marked with * are significant at P < 0.05, ns = not significant.

Degree
interiors than in the edges of most fragments under study.
Similarly, the percentage contribution of MBN to TKN was highest in the interior microsite of Very Large followed by Medium, Large and Small fragments (Table 6).The percentage contribution of MBP to Ex. P was high at the edge in the Very Large (5.22%) followed by the Large (4.45%), Medium (3.55%) and Small (3.41%) fragments.A similar trend was observed in the forest interior with the highest contribution by the Very Large (4.70%) and the lowest by the Small fragment class (3.48%) (Table 6).

Discussion
Fragmentation of the forest resulted in considerable changes in the microclimatic variables, soil properties and microbial biomass.The low values of nitrogen in the edge could be the inability of the plants to uptake nitrogen from the soil after disturbance and also due to leaching losses (Jose et al., 1996;Ruitta et al., 2012).Inorganic-N (NH 4 + -N and NO 3 --N) concentrations were lower in the edge microsites and Small fragments.However, Ex.P showed no consistent trend along a fragment size gradient.This finding is similar to that observed from tropical deciduous forest of Mexico (Toledo-Aceves & Garcia-Oliva, 2008).Soil nutrient availability depends on abiotic and biotic factors such as mineralization, plant uptake, microbial uptake and leaching losses.Alteration of microclimatic conditions due to edge might have affected various nutrient return processes such as litter decomposition rate (Ruitta et al., 2012) microbial decomposition activities (Lazaro-Nogal et al., 2012) thereby causing the variation in nutrient content among the microsites.
Habitat degradation due to human disturbances and fragmentation reduce canopy cover and initiates microclimatic variation in the remnant fragments (Brothers & Spingarn, 1992;Chen et al., 1999).The higher microbial biomass -C, -N and -P in the larger fragments and interior microsites (P< 0.05) of the forest fragments can be attributed to an increase in light intensity and a decrease in air temperature, relative humidity, soil temperature and soil moisture content as well as soil organic carbon and nutrient (TKN and Ex.P) content.Similar results were found in tropical deciduous forest where microbial biomass increased from edge into the interior of forest fragments (Toledo-Aceves & Garcia-Oliva, 2008).The low soil microbial biomass at the edges is associated with low SOC, ) SOC (μg g -1 ) TKN (μg g -1 ) ExP (μg g -1 ) MBP (μg g -1 TKN and Ex.P as compared to the interior.Similar relationships between SOC, TKN and Ex.P with microbial biomass have been observed in temperate forests of Spain (Diaz-Raviña et al., 1993) and subtropical forests of Meghalaya (Kamei, 2007).Small fragments were prone to disturbances due to easy accessibility and dominated by edge environment (Pao & Upadhaya, 2017).This might have led to low microbial biomass in the smaller fragments.
The highest values of microbial biomass -C, -N and -P in the present study during winter season coincide with low air and soil temperatures (12.3 ºC and 11.5 ºC respectively).It also indicates the period of low microbial activity and greater nutrient immobilization in the soil microbial biomass.In contrast, lower values of microbial biomass observed during rainy or autumn seasons when temperature and soil moisture conditions were favourable for the growth of microbes indicated the period of rapid turnover and cell lysis contributing to the mineralization of nutrients in soil (Singh et al., 1989).This finding is similar to that observed in seasonally dry tropical forests and savanna of the Vindhyan region of India (Singh & Kashyap, 2006).Greater demand of nutrients by plants during rainy season when majority of them are at their peak vegetative growth, further limits the availability of nutrients to soil microbes, thereby reducing their immobilization in microbial biomass (Singh et al., 1989).Seasonal variations observed in microbial biomass-C, -N and -P in the present study is in agreement with other studies from subtropical forests of northeast India (Upadhaya et al., 2006;Kamei, 2007) and China (Wang et al., 2016).
Microbial organic content stoichiometry (MBC: MBN, MBC: MBP) were higher in the edge.In Small fragments, the higher MBC: MBP ratio in the edge could be attributed to immobilization of phosphate to cope up with reduced P availability.The mean C/N ratio (10 -12) of soil microbial biomass in the studied forest fragments is closer to the range (6.5 -10) reported from subtropical natural secondary forest (Chen et al., 2013;Xing et al., 2010), and subtropical forest (9.4) of Australia (Burton et al., 2010).The values in the present study indicate that the soil microbial communities in the fragments were dominated by the fungal population (Brume & Khanna, 2009).The microbial biomass C/P ratios obtained in the present study (16 -19) is close to that (18 -26) reported from subtropical (Devi & Yadava, 2006) and tropical (Barbhuiya et al., 2008) forest of the region.
Microclimatic factors and soil properties in the fragments played an important role in influencing microbial biomass (Table 7).The present study showed that microbial biomass -C, -N and -P was influenced by soil -moisture content (r = -0.77,-0.77, and -0.76 respectively) and -temperature (r = -0.65,-0.87, and -0.65 respectively, P< 0.01).With the onset of monsoon, the soil gets saturated and during this period the temperature is also high.Such conditions favour the growth of microbial population.However, during this period there is a rapid turnover of microbes due to lysis, microvory and high demands of nutrients by the plants (Singh et al., 2010) leading to low microbial biomass.
Our result suggests that soil microbial biomass increased along a fragment size gradient and was influenced by the prevailing microclimatic conditions and soil properties.Changes in soil microbial activity may alter the interaction of plant, soil and microbial community that would gradually degrade in the fragments due to edge effect.Such changes may become unstable and unpredictable in fragments that are exposed to human disturbances.Therefore, conservation of soil biological processes which is a key to nutrient cycling is essential for the stability of ecosystem functions in fragmented landscapes.
class.The ratios for the different fragment classes with their respective microsites viz., edge and interior were 11.05 and 10.29 in Values are mean of two years ± SE (n = 18); A= autumn, W = winter, S = spring and R = rainy; E = Edge, I = Interior).

Table 1 .
Vegetation characteristics in the studied fragments.

Table 2 .
Soil physical properties in the studied fragments.

Table 3 .
Seasonal fluctuations of microenvironmental variables and soil properties in the edge and interior of the studied fragments.

Table 4 .
Two-way analysis of variance (ANOVA) showing the effects of fragment size class, microsites and seasons on microenvironmental variables and soil properties.

Table 6 .
Mean MBC, MBN and MBP (μg g -1 ) and its proportion relative to SOC, TKN and Ex.P.