Ecology and Diversity of Diatoms in Kuttanadu Paddy Fields in Relation to Soil Regions , Seasons and Paddy-Growth-Stages

This assessment of ecology and diversity of Diatoms in Kuttanadu, is continuation of the previous publications of Green-algae and Blue-green-algae of the same region; all the three investigations were carried out simultaneously. The unique Kuttanadu wetlands (90 ̊17'N to 90 ̊40'N; 760 ̊19'E to 760 ̊33'E), a well known ‘Rice bowl’ of Kerala, spread over 53,639 hectares, is located in Alapuzha District of Kerala, India. This wetland was once known for its high biodiversity, but currently severely affected by intensive green-revolution activities of past few decades. Main objective of the present report is to explain the diversity and ecological status of Diatoms in relation to the environment conditions currently existing in the region. Duration of the study was from December 2009 to October 2010. Soil samples of three different soil-types in two different crop-seasons, at two diverse crop-growth-stages are analyzed and compared. Altogether 120 composite soil samples randomly collected from the broad wetlands are analyzed. A rich community of Diatoms, of 40 species is found out. The order Naviculales is observed as the dominant Diatom (40%) in these paddy wetlands. Ecological parameters of Diatoms were found highest in the Lower Kuttanadu soil region, during Virippu season, at the seedlingstage of the crop, whereas the lowest value for most of the parameters were observed in Kayal soils during Puncha season at the seedling-stage. Apart from the specific soil factors influencing Diatom population characteristics in the region, crop seasons and soil phosphorus are found to have significant influence on Diatoms in this wetland paddy soils.


Introduction
This is the third in the series of reports on ecology and diversity of algae of Kuttanadu wetlands, the first two parts, one on green-algae and the other on blue-green-algae are already published (Vijayan & Ray, 2015a, 2015b).
Diatoms are one of the most diverse groups of algae, estimated to be more than 100,000 species, with cosmopolitan distribution (Demirbas & Demirbas, 2011).Uniqueness of Diatoms is that they are not just universally abundant group of algae, but are highly ecologically diverse (Jena, Ratha, & Adhikary, 2006).Moreover, they are one of the most successful groups of photosynthetic eukaryotes (Muruganantham, Gopalakrishnan, Chandrasekharan, & Jayachandran, 2012).Seasonal change of diatom community in flooded paddy soil environments is a widely discussed topic worldwide.
Another major significance of the study of Diatoms is that they are well known bio-indicators, capable of providing specific information on varieties of specific environment characteristics (Seirieyssol, Chatelard, & Cubizolle, 2010) of specific habitats.Some of them are highly tolerant to harsh environmental conditions, while some others are highly sensitive to soil conditions such as desiccation, freezing and abrupt heating (Souffrean, 2010).Such characteristics make the group ideal ecological indicators (Mazumder, 2012).Like all other algae, diatoms also contribute significantly to carbon sequestration; but the role of diatoms in the recycling of silica is highly specific (Kesic, Tuney, Zerben, Guden & Sukatar, 2013).Diatoms constitute a fundamental link between primary and secondary production in aquatic systems.Therefore, the major ecological importance of diatoms is as a component in the food chains of specific aquatic species.Influence of diatoms on (deep black in colour with high proportion of organic matter, characterized by acidity and salinity found mainly in the Lower Kuttanaduand, and rarely in some parts of the upper Kuttanadu zones) and 'Kayal' (the reclaimed shallow beds of Vembanadu Lake, mainly silt-loam to silt-clay-loam) soils (Pillai, Ponniah, Vincent, & David, 1983).All the geographic and other details of the three soils regions are well described in the previous reports (Ray, Dhanya, & Binoy, 2014).The Kuttanadu is a unique wetland ecological zone with high biodiversity, especially of indigenous fishes, but quite destroyed by intensive green-revolution efforts of past few decades in the area.It is believed that once up on a time, the brackish water environments of Kuttanadu promoted a rich diversity of mangroves and provided ideal habitats for diverse flora and fauna.

Soil Sampling Procedure
Soil samples were systematically collected from all the three soil regions in two different paddy cultivating seasons of a year; in each season, samples of two different paddy growth stages were collected.At each site two broad specific paddy fields located at 1-2 km intervals were randomly selected for sampling.In each specific field, several plots (5-10) of about 10m 2 areas were randomly identified for sampling.Several soil samples (5-10 from a plot) from the upper 1-5 cm soil layer from all these plots were collected and put together in a sterile cotton bag using aseptic implement.Repetitive samples put in each cotton bag from different plots of a single paddy field on air drying were mixed thoroughly to a composite sample.There were five sites in a soil region.Since there were five sites, two sampling fields for each site and two crop seasons (the Puncha -summer crop of December 2009 to March 2010; the Virippu -the additional crop of June 2010 to October 2010) to sample and two crop-growth stages (seedling stage and panicle stage) at each season to sample, altogether, 40 composite soil samples (5 x 2 x 2 x 2) were collected from each soil region.Altogether, there were 120 composite soil samples (3 x 40) representing the three soil regions of Kuttanad -the 'Upper Kuttanadu' or 'Karappadom', the 'Lower Kuttanadu or 'Kari'' and the 'Kayal' zones for the soil study and for diatom studies.

Study of Diatom Flora
Methods of Van de Vijver and Beyens (1998) and Jena et al. (2006) were used in the microscopic examination of soil samples for diatoms.Microphotograph of each specimen was taken using Olympus digital Camera attached to light microscope.Taxonomical descriptions of Gandhi (1956), Sarode and Kamat (1984), Taylor, Archibald and Harding (2007) were followed in the identification of species.

Scanning Electron Microscopic (SEM) Analyses of Diatoms
Soil samples for diatom analyses were placed in air tight plastic bags to prevent moisture loss from the algae and the samples were repeatedly sprayed with iodine to preserve them.The samples were then sieved through a 75 micron nylon mesh.The SEM studies of cleaned specimens were carried out after ion coating in high vacuum mode Jeol make model-JSM 6390 LA machine.The specimen were fixed to carbon tapes (1 x15 cm) placed on aluminium stubs, coated with gold and underwent magnetron sputtering by auto fine coater -Jeol JFC-1600.The accelerating voltage was 20KV.SEM analyses were carried out in the laboratory of the sophisticated test and Instrumentation Centre (STIC), University of Science and Technology, Cochin.

Environment Relationships and Population Dynamics of Diatoms
Correlation of diatoms to environmental factors such as region, season, and physiochemical soil parameters are described.Method of Dey, Tayung and Bastia ( 2010) is followed in the study of relative abundance of species in the community.Diversity index (Shannon Wiener Index) and species richness of all species of diatoms in different seasons were worked out as per Shannon & Wiener, (1949).Species richness was calculated following the method of Whittaker, (1977).Classification of taxa was done as per the classification of Guiry & Guiry, (2012).

Results
Altogether 40 species of diatoms ( Order: Melosirales

Season
The highe observed a species (2 'Virippu' c The most that of 'Ka abundant s

Ecolog
The Sps. Rich -species richness; Div.Ind -Diversity Index.Ray et al. (2014) Variations in pH, total organic carbon (C), total/ Kjeldal nitrogen (N), plant available phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) studied in relation to crop seasons and different stages of paddy growth in the three different regions of Kuttanadu are given in Table 4.

SEM Analyses of Soil Samples for Diatoms
SEM analyses of the Kuttanadu soil samples revealed the presence of seven species of diatoms (Figure 5).Randomly selected six soil samples from the three regions of Kuttanadu were analyzed for diatoms.

Discussion
Soil is a complex heterogeneous environment (El-Gamal, 2008).Human influences affect its physical, chemical, and biological characteristics.In general, high diatom diversity is used as an indicator to anthropogenic disturbance and environmental contaminations (Vacht, Puusepp, Koff, & Reitalu, 2014).Among algae, diatom communities are considered as indicators of both organic and anthropogenic pollutions.In the present study, diatom diversity was reported to be high in almost all regions, seasons and growth stages, which indicated that the disturbed nature of this agro-ecosystem.In the current investigation, the highest number of diatom species, total isolates, species richness and diversity index were observed in the Lower Kuttanadu paddy soils in the seedling stage (early monsoon or pre-monsoon) of Virippu cultivation, which gradually decreased towards panicle-growth-stage (the monsoon season).
Comparatively lower diatom diversity is observed in non-monsoon crop season (Puncha cultivation) than in the monsoon crop (Virippu), which showed the influence of climate in determining the diatom density of Kuttanadu wetland paddy soils.Ecological conditions have prominent role in determining the algal diversity (Lin, Chou & Wu, 2013).Redekar and Wagh (2000), Pareek et al. (2011), Muralidhar andMurthy (2014), and Jayabhaye (2010) noticed that diatom diversity reaches maximum during monsoon or pre-monsoon months and minimum during summer.Contradictory to these findings, Muruganantham et al. (2012), observed maximum diversity of diatoms in summer.
According to Metting (1981), Neustupa and Skaloud (2008) and Lames -Da-Silva, Branco & Necchi-Junior, (2010) light play a significant role in the growth and differentiation of algae.In the present study, highest diatom diversity and abundance was observed in seedling stage of second cultivation (Virippu), when light is abundant due to less canopy of the crop plant.Roger and Reynaud (1982) also observed that diatom community increases in paddy fields during seedling stage of the crop.Lin et al. (2013), Fujita andOhtsuka (2005), also supported this view that diatom cell density reaches the highest number just after transplanting of rice and thereafter decreases.
In the current investigation, the Diatom species richness and diversity index were found to be correlated (Pearson's correlation coefficient) to soil parameters such as pH, N, P, K, Ca and Mg of different regions of Kuttanadu at different crop-growth-stages and crop-seasons.A variety of physical and biological factors influence the distribution and diversity of diatoms.Comerton and Houghton (1978) observed that inorganic nutrients stimulates or suppress algal growth in the soil.Nutrient concentration is the most important factor that influences the distribution and abundance of Diatom species (Broady, 1979;Mohan, Shukla, Patil, Shetye, & Kerkar, 2011).In the present study, highest Diatom diversity was observed in 'Lower Kuttanadu' soil.Soil factors, vegetation and topography affect the algal biomass (Patrick, Groffman, Driscoll, Fahy, & Siccama, 2001), especially diatoms (Venkitachalapathy & Karthikeyan, 2013).Soil of 'Lower Kuttanadu' region is clay loams characterized by a fairly high amount of nitrogen.Diatom diversity, community behaviour and composition are influenced by nitrates (Muralidhar & Murthy, 2014).But in the present study, nitrates in Kuttanadu soils are not found to be correlated to any diversity parameters of Diatoms there.On the other hand, species richness and diversity index showed a significant positive correlation to phosphorus.This is in agreement with Comerton and Houghton (1978) that phosphorus increases the algal count, especially that of Diatoms in natural environment.
In the present study, species richness and diversity index showed significant negative correlation with pH.This is in agreement with the observations of Muralidhar and Murthy (2014) that Diatoms are sensitive to pH and salinity.Lukesova (2001) observed that Diatoms prefer neutral to alkaline pH.But, Diatoms showed high diversity in Kuttanadu soils, where pH ranges from 3.4 to 4.6, which is in agreement with the observations of Muralidhar and Murthy (2014) that pH has no role in the periodicity and density of Diatoms in soils.

Conclusion
Overall, the present analysis of the ecological characteristics of Diatoms of Kuttanadu wetland paddy fields in relation to soil regions, crop-seasons and paddy-growth-stages appears quite useful to the exploration of the economic potentials of many individual species of Diatoms.Since many of the diatoms observed in this investigation are found quite specific to soils or seasons or crop growth stages, further investigations on the environment factors contributing to limited occurrence of them in specific locations alone, would enable identification of them as bio-indicators of diverse environmental conditions.
is carried out after pumping out the excess water of the broad fields, which during the season of paddy cultivation remain separated from the surrounding backwaters by artificially reclaimed protective 'barrages'.Majority of the fields are cultivated twice a year.The non-monsoon crop, 'Puncha', is the main crop, which is sown in November or December and harvested by the end of March.The additional monsoon crop, 'Virippu' is sown in May to the end of June and harvested in September or October.Upper Kuttanadu, Lower Kuttanadu and Kayal lands are the three soil regions of Kuttanadu.Three kinds of soils reported from the area are 'Karappadom' (river-borne alluvial soils found mostly in the Upper Kuttanadu), 'Kari'

Table 1 ;
Figures 2 & 3)belonging to 19 genera have been recorded from Kuttanadu paddy fields during the entire study period.Diatoms collected belonged to nine orders,

Table 3 .
Minimum diversity index (1.06) was noticed in 'Kayal' lands during 'Puncha' cultivation season at seedling-growth-stage of the crop.The species evenness showed very slight variation from 0.315 -0.317 in different regions of Kuttanadu during the two crop seasons and crop-growth-stages.Species richness and diversity index of diatoms in different seasons and vegetations (UK -upper Kuttanadu; LK -lower Kuttanadu; KL -Kayal lands; R1-Upper Kuttanadu, R2-Lower Kuttanadu and R3-Kayal