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Changes in pmoA gene containing methanotrophic population and methane oxidation potential of dry deciduous tropical forest soils

In natural ecosystems such as forests topographical gradients, species composition variability and seasonality, are the potential drivers of methane (CH4) metabolism, and thus, of the population size and activities of methane oxidizing bacteria (MOB). To test the hypothesis that topography, tree species and seasonal variability influence MOB population and soil methane oxidation potential (MOP), we conducted two consecutive years of study selecting three sites in a dry deciduous tropical forest in Chandauli district of eastern Uttar Pradesh, India. The qPCR results showed a large variation in MOB population size (copies g dws), ranging from 1.0 × 10 to 2.1 × 10 and 9.0 × 10 to 2.2 × 10 during 2016 and 2017 respectively. The distribution of MOB population revealed the trend: hilltop > middle > hillbase with its maxima in the winter and minima in the rainy season. Laboratory incubation study revealed a similar trend in soil MOP (ng CH4 g h dws), it varied from 10.6 to 20.6 and 10.5 to 20.7 during 2016 and 2017 respectively. The outer canopy soils showed lower MOB population and MOP compared to under canopy soils of tree species Butea monosperma, Madhuca indica and Tectona grandis during both years of study. The topography, season and tree species significantly influenced the MOB population size and MOP. Soil MOP showed a highly significant correlation (r = 0.89; p < 0.01) with MOB population, and a negative correlation was found with soil moisture (r = 0.76; p < 0.01). The results indicate that the dry deciduous tropical forest soils are potential sinks of atmospheric CH4 wherein, the MOB population characteristically responds to topographical changes, tree species and seasonal shifts driving collectively the overall MOP of forest soils.

the beginning of the industrial era (1750) to 2011, CH 4 concentration has increased from 715 to 1803 ppb; with 1% annual increase over a century 2 . The largest sink (about 90% of the total CH 4 ) is its photochemical oxidation mediated by reactions with hydroxyl (OH -) radicals 3 . Other significant sinks include diffusion into the stratosphere, and microbial oxidation. Although total CH 4 sink strength in terrestrial ecosystems is driven by microbial oxidation in the order 33.5 ± 0.6 Tg CH 4 /y (ref. 4), role of the latter cannot be underestimated as these are important contributors to attenuate CH 4 flux at the oxic-anoxic interfaces 5 .
Methane oxidation in aerobic soils is mediated by methanotrophs (methane oxidizing bacteria; MOB), a subgroup of methylotrophs. These bacteria utilize CH 4 as the sole carbon and energy source 6,7 . Among terrestrial ecosystems, methanotrophic community inhabiting forest soils has been identified as a major contributor to atmospheric CH 4 oxidation 8 . Atmospheric CH 4 oxidation studies are mainly confined to temperate forest soils 9-11 , whereas similar reports in tropical and subtropical forest soils, are very limited [12][13][14] . Some earlier studies suggested that CH 4 oxidation potential (MOP) of soils depends on the diffusive gas transport 8 , soil nitrogen content 15,16 , land use 17 , soil temperature 18 , pH 19 , salinity 20 , season 21 , topographic positions 22 and tree species 23 . The CH 4 uptake rate studied in temperate forests reported higher values during summer compared to winter 21,24 . In earlier studies, soil moisture and temperature were the key regulatory factors that drive CH 4 oxidation in soil. High soil moisture creates barrier for oxygen diffusion into the soil, required by aerobic methanotrophic bacteria, whereas increasing temperature up to optimum level (25-35°C) enhanced the methanotrophic activity 25 . Methanotrophic population and their activity are also influenced by soil physical properties 26 . Thus it can be assumed that multivariate factors are linked to the regulation of CH 4 oxidation in the natural ecosystem, however, the pattern of MOB population and their activity are not well documented. Most studies in forest soils are, hence focused on the activity and diversity of methanotrophs. For tropical dry deciduous forest soils, the information on MOB population size seems equally important but has not been studied well. Quantitative data in these lines can be used to relate potential changes in gene abundance and/or the level of gene expression associated with shifts in abiotic determinants 27 . Accordingly, systematic database on the magnitude of MOB population size may possibly add to furthering our understanding of the significance of MOB in the ecology of CH 4 oxidation in forest soils. Few reports are available on the population size of methanotrophs in forest soils of temperate regions 28,29 . Giri et al. 30 used most probable number (MPN) technique for counting MOB in tropical forest soils. This technique, however, has its own bias due to selective culture conditions. Overall, in tropical forest soils, information on MOB is almost scanty, particularly in the context of Indian tropical forests that constitute a major global share 31 .
Understanding the key determinants of activity and MOB population size here, has relevance because the dry deciduous tropical forest of Vindhyan region constitutes ~53% of total forest area of India. This study was targeted to investigate the effect of topography, tree species and seasonal factors on MOP and MOB population size in the dry deciduous tropical forest soils of dry tropics.

Study site
The experimental sites were located in the dry deciduous tropical forest of Vindhyan plateau situated in Chandauli district of eastern Uttar Pradesh, India. The climate of this region is tropical monsoonal, and receives about 82-85% rainfall during rainy season (late June-September) due to southwest monsoon. The whole year is divisible into three seasons: rainy (late June-September), winter (October-February) and summer (March-early June). The annual mean rainfall ranges from 400 to 900 mm, while mean monthly temperature varies between a minimum of 8°C in January to maximum 42°C in June. The average rainfall in rainy, winter and summer season during study period was 685.0, 3.0 and 2.0 mm respectively. The soil order of the study sites was Ultisols and red to brown in colour. The vegetation in the study area represents northern tropical mixed deciduous forest 32

Experimental design and soil sampling
Soil samples were collected from three forest sites of Naugarh, Jamsoti and Chakia, representing upper, middle and lower elevation landscapes at a height of 300 m, 150 m and 90 m respectively (Table 1). At each site, three plots of 1 hectare (ha) area were randomly demarcated, based on the respective dominance of Butea monosperma, Tectona grandis and Madhuca indica. From each plot, soil samples were taken from 0-15 cm depth using a corer in triplicate at five randomly selected locations. Triplicates were mixed to form a composite sample (n = 15). Also, at each site, three plots, 1 ha each, were selected to represent off-canopy condition. Thus, a total of 30 composite soil samples (15 each for inside and outside canopy) were collected from each site in a month, and three times in a season (rainy: July, August, September; winter: November, December, January; summer: March, April, May), for two consecutive years (2016 and 2017). Soil sampling was avoided during February, June and October as they were identified as transition month of winter-summer, summer-rainy and rainy-winter seasons respectively. Soil samples were sieved (2 mm mesh) to remove fine roots, and were aliquoted into two: one part was stored at -20°C for qPCR analyses and the other at 4°C for CH 4 oxidation experiments and soil physico-chemical studies. All analyses were done in triplicate.

Soil properties
Soil variables such as pH, texture, bulk density (BD) and water holding capacity (WHC) were determined using standard protocol described in Vishwakarma and Dubey 33 . In brief, the soil particle proportions were estimated using sieves of variable mesh sizes and hydrometer method 34 . The BD of the soil samples were determined by core method 35 . The perforated circular brass boxes were used to determine WHC of the soil samples as described by Piper 35 . The soil : water (1 : 2.5; w/v) suspensions were used to measure soil pH using pH meter equipped with a glass electrode (CyberScan pH 510, Eutec Instruments Pte Ltd). The electrical conductivity was measured in soil water suspension using digital conductivity meter (Systronics, India). Macrokjeldahl method was used for total soil N estimation 36 . For the measurements of organic carbon (C), dichromate oxidation procedure was followed 37 . Leaf litter was collected during the peak litter fall period (February to March) of the year 2017. The litter samples were oven dried, and powdered to determine C and N content as per the standard protocol.

Soil moisture and ammonium-N content
Soil moisture was determined by gravimetric method. Exchangeable NH + 4 -N was estimated in 2 M KCl extracts, and analysed spectrophotometrically by phenate method 38 .

Methane oxidation potential
Laboratory incubation experiment was used to determine CH 4 oxidation in soils 33 . In brief, 10 g of soil sample was allowed to equilibrate for 24 h at 25°C, and then, transferred to gastight 135-ml Erlenmeyer flask, added with 50 μmol l -1 (~1150 ppmv) CH 4 (Sigma Aldrich; 99.9% pure), and incubated for 10 days in dark at 30°C. Further, for CH 4 analysis, headspace samples (100 μl) were withdrawn using a gas-tight glass syringe every 24 h up to 240 h. The gas samples were analysed in Agilent 7820A gas chromatograph attached with a flame ionization detector (FID), Porapak N column, and nitrogen as the carrier gas (flow rate 12 ml min -1 ). The temperature for column, injector and detector was maintained as 180°C, 210°C and 250°C respectively. MOP is expressed as nano gram CH 4 per hour per gram of dry weight soil (dws).

Population size of MOB
Genomic DNA was extracted from the forest soil samples (0.5 g) using FastDNA Spin Kit for Soil (MP Bio-medicals, LLC, Solon, Ohio, USA) by a bead beater (FastDNAprep, MP Bio, USA) for lysis of cells according to a manufacturer's protocol. Real-time PCR was performed using iCycler iQ5 thermocycler (Bio-Rad). Population of methanotrophs was quantified using specific primer pair A189f 5′GGNGACTGGGACTTCTGG3′ and mb661r 5′CCGGMGCAACGTCYTTACC3′ (refs 39, 40). The reaction mixture (20 μl) contained 10 μl of PowerUp™ SYBR™ Green Master Mix (Applied Biosystem), 0.4 μl of each primer (10 μM), 2 μl DNA template (50 ng/μl) and DNAase/RNase-free water to the final volume of 20 μl. For each sample, the reactions were performed in duplicate and for plasmid standards, samples were analysed in triplicate. PCR conditions for methanotrophs were optimized as three step cycling protocol with the initial 4 min denaturation (94°C), followed by 40 cycles of denaturation (94°C for 15 s), annealing at 56°C for 30 s and extension at 60°C for 30 s. Fluorescence data were acquired at 77.5°C after each consecutive cycle. After completion of 40 cycles, the melting curve analyses were performed by increasing the temperature from 60°C to 95°C and recording fluorescence for every 0.5°C increase. Plasmid DNA containing the target gene (methanotrophic bacteria specific pmoA gene) was taken as the standard DNA in PCR (real time) assay acquired by PCR-cloning using A189f and mb661r primer pairs. Plasmids were isolated using HiPurA (TM) Plasmid DNA Miniprep Purification Kit (Himedia), and DNA concentration was determined by Nanodrop 2000 spectrophotometer (Thermoscientific). Standard curves were prepared with triplicate 10-fold dilutions of plasmid DNA 29 .
Data were tested for PCR efficiency. The values for methanotrophs (slope: -3.729; R 2 : 0.983; efficiency: 85.4%) were within the suggested range. Zhang and Fang 41 mentioned that in practice, for an appropriate standard curve, R 2 should be above 0.95, with slope from -3.0 to -3.9 corresponding to PCR efficiency of 80-115%.

Statistical analysis
For statistical treatments, data were subjected to multivariate analysis of variance (MANOVA) to record significant effect of year, topography, season and tree species on the observed variables. Simple Pearson's correlation coefficient analysis was applied to determine the relationship of parameters to each other. The statistical analyses were performed using IBM SPSS version 20.0 (ref. 42).

Soil properties and leaf litter C and N
Results on soil characteristics are presented in Table 1. Soil texture consisted predominately of silty type at higher topography (Naugarh and Jamsoti), while silty clay loam type at hillbase (Chakia). Bulk density varied from 1.77 to 1.94 g cm -3 . The bulk density was highest at hilltop followed by middle and hillbase. The soil pH ranged from 5.21 to 7.53. The water holding capacity was maximum for hillbase followed by middle and hilltop. Total organic N and C ranged from 823.6 to 1536 and 6220.7 to 16311.0 mg kg -1 respectively, with highest value at hillbase followed by middle and hilltop. The EC value ranged from 21.7 to 74.96 μS m -1 along the topography. In leaf litter samples, C content was maximum (467.5 mg g -1 ) in T. grandis followed by M. indica (433.07 mg g -1 ) and B. monosperma (413.07 mg g -1 ). The N content also followed the similar trend as C with the value 20.1, 10.3 and 5.1 mg g -1 for T. grandis, M. indica and B. monosperma respectively.

Soil moisture and ammonium-N content
Soil moisture and NH + 4 -N content at each site during the study period (2016 and 2017) have been shown in Table  2. Soil moisture was significantly higher during rainy season, and it ranged from 11.9% to 22.4%, 3.7% to 9.8% and 3.6% to 9.4% during rainy, winter and summer season respectively, at all the sites throughout study period. At all the sites, soil NH + 4 -N content varied from 0.07 to 0.76, 0.46 to 1.01 and 0.55 to 1.05 μg g -1 dws during rainy, winter and summer season respectively. The NH + 4 -N content of the soil showed pattern reverse to that of soil moisture being maximum during summer, followed by winter and rainy season at each study site. The significant effect of the topography (F 1,214 = 900.88; p < 0.001), season (F 2,213 = 1864.24; p < 0.001), tree species (F 3,212 = 82.34; p < 0.001), except year (F 1,214 = 0.76; p > 0.001) was observed on the soil NH + 4 -N content.

Quantification of MOB population
Variations in methanotrophic population size along the topography, in different seasons, and tree species for the year 2016 and 2017 are depicted in Figure 2. The population size of methanotrophs as estimated by the pmoA gene copy number varied between 7.0 × 10 5 and 3.0 × 10 7 copies g −1 dws. The MANOVA showed significant effect of topography (F 2,213 = 63.58; p < 0.001), season (F 2,213 = 584.52; p < 0.001) and tree species (F 3,212 = 41.5; p < 0.001) on the population size of methanotrophs. The population size was smallest in soil samples from the outer canopy. The methanotrophic population size was maximum in soils from the under canopy of Butea monosperma, followed by Madhuca indica and Tectona grandis irrespective of the topography and season.

Effect of topography
Methane oxidation range recorded here (9.9 to 21.5 ng CH 4 g -1 h -1 dws), is comparable with those observed for soil of the temperate forest in Massachusetts 43 , hardwood forest of South Korea 44 , and the deciduous forest of Europe 29 . Topographical gradient showed significant (p < 0.001) effect on CH 4 oxidation being highest at hilltop followed by middle and hillbase site. Such a difference in CH 4 oxidation values could be correlated with high population size of methanotrophs at higher topography followed by middle and hillbase ( Figure 2). So far, MOB population in terms of gene copy number is not available for data comparison from other regional scale studies. The data presented here, which form the first report from Indian tropics, can be used for comparing the values reported from other ecozones/ecosystems using comparable methodologies. Barcena et al. 28 reported MOB population between 10 6 and 10 7 copies g −1 dws in oak dominated temperate forest of Denmark. Malghani et al. 29 recorded methanotrophic population ranging from 4.3 × 10 7 to 7.5 × 10 7 copies g -1 dws in a temperate deciduous beech forest of Germany. We found strong correlation (r = 0.89; p < 0.01) between CH 4 oxidation and methanotrophic population irrespective of the topographic variation. Further, the hilltop site showed significant negative correlation (r = -0.76; p < 0.01) between MOP and the soil moisture content. The results of regression analysis of CH 4 oxidation and methanotrophic population to the soil physicochemical variables (BD, clay content,  c and d), and hillbase (Chakia) (e and f ) study site for two consecutive years. Different uppercases denote significant differences (at p < 0.01) between tree species in the same season and different lowercases denotes significant differences between seasons in the same tree species according to the Tukey's HSD test.
WHC, total organic carbon, total nitrogen and electric conductivity) are presented in Table 3. The clay content of the soil was found to be negatively correlated with the CH 4 oxidation and MOB population size (R 2 = 0.98; p < 0.001). The hilltop site with high coarse particles showed higher CH 4 oxidation compared to hillbase rich in clay content. This result is similar to that of Boeckx et al. 45 who reported higher CH 4 uptake rate in coarse soil compared to fine textured soils of Belgium forests. Fine particles of the soil regulate CH 4 uptake by regulating the diffusion of atmospheric CH 4 and oxygen in the soil 46 . In general, the site situated at the lower elevation, receives high erosional residues rich in soil organic matter, soluble salts, mineral colloids, etc. subject to natural downhill movement assisted by runoff. Subject to limited O 2 supply, the accumulation of organic substances creates anaerobic microhabitats and consequently limits methanotrophy 47 . The soil organic carbon and nitrogen were found to be negatively correlated with CH 4 oxidation and population size ( Table 3). The overall results show that topographical variations with characteristically different soil properties are important determinants of population size of CH 4 oxidizers, and consequently the MOP of the soil.

Effect of tree species
MOP and MOB population size, both varied significantly with tree species. The soil samples from the under canopy of Butea monosperma showed maximum CH 4 oxidation rate followed by those cultivated with Madhuca indica, Tectona grandis and outer canopies. The outer canopy soil with poor organic input was poor in CH 4 oxidation. The methanotrophic population at each outer canopy site, during every season was also poor. In a previous study, Reay et al. 23 reported the effect of vegetation type (alder, oak, Norway spruce, Scots pine and grassland) on high and low affinity MOP in Bowland forest, UK. They reported large reduction in CH 4 oxidation in alder soils than those from other vegetation types, and linked the process with the inhibitory effects of high nitrate concentrations. Tree species influence nitrogen capture and mineralization 48 . The litter composition strongly influences microbial colonization. The organic C and N content of the leaf litter of the tree species considered in this study showed significant variations. Fresh leaf litter of Tectona grandis, rich in organic C and N, might release N to the soil in proportion high enough to interfere with CH 4 oxidation through regulation of methanotrophic activity. Soil moisture also varied significantly (p < 0.05) with tree species, being highest for the under canopy soil of T. grandis followed by M. indica and B. monosperma ( Table 2). The CH 4 oxidation rate and MOB population size were negatively correlated with the soil moisture (Table 4). Therefore, variation in soil moisture content could lead to changes in CH 4 oxidation across under canopy soils of different plant.

Effect of season
The MANOVA revealed significant (p < 0.001) seasonal variation in CH 4 oxidation. Seasonal variations in CH 4 oxidation were well linked with soil moisture. During rainy season, with maximum soil moisture, CH 4 oxidation declined sharply (Figure 1). Previous reports also indicated decrease in CH 4 oxidation rates 21 with increasing soil moisture. The population size of methanotrophs was high during winter, followed by summer and rainy season ( Figure 2). In summer, the moisture content of the soil goes below 5%, which adversely affects the microbial population. Although soil moisture content is negatively correlated with CH 4 oxidation, it may show positive correlation below 8% (ref. 49). Ammonium-N was maximum in summer, followed by winter and rainy ( Table 2). High ammonium-N content in the soil might be attributed to low nitrification during summer. In rainy season, high demand for nutrients by the growing vegetation leads to decline in NH + 4 -N (ref. 50). While in most cases, CH 4 oxidation of in forest soils is inhibited by N additions 51,52 , NH + 4 -N in our study, showed significant but poor positive correlation with CH 4 oxidation ( Table 3). Methane oxidation is inhibited in case inorganic N was substantially high. Positive effects of N on CH 4 oxidation have been reported in rice field 53 and forest soils 54 .

Inter-annual variation
The CH 4 oxidation and methanotrophic population did not show significant annual variation although, for soil moisture it was significant. The inter-annual variation in soil moisture might be due to relatively poor rainfall during 2017. Although methanotrophic population showed negative relationship with the soil moisture, no significant changes were recorded annually.

Conclusions
The estimates of MOP of dry tropical forest soils provide a reference for comparing other ecosystems in terms of process and the associated microbial communities. Our analyses document the extent of variation in MOP and MOB population distribution in relation to ecosystem variables linked with topography, tree species and seasonality. The soils of all the forest sites showed CH 4 oxidizing potential, although it varied significantly with the topography, season and tree species. The soil MOP showed strong positive correlation with the MOB population size. Soil moisture emerged as the important driver regulating CH 4 oxidation largely by regulating the diffusion of CH 4 and O 2 . The study unravels the drivers of CH 4 cycle in the seasonally dry tropical forest, and will certainly help in exploring regional contributors to global CH 4 cycle. The study merits adding new dimensions furthering the need for assessment of molecular diversity of MOB, establishing the causal linkages, and enhancing sink efficiency of forest soils.

Conflict of interest:
Authors have no any conflict of interest for this publication