Millipede Species Enrich the Fertility of Soil in Alagar Hills Reserve Forest

Soil biota can interact with litter decomposition and significantly change nutrients of soil through egestion of faecal pellets. In the present study, examined the interactions between soil fertility and millipedes occurrence in Alagar hills Reserve forest. Millipedes as detritivores significantly affect nutrient cycling through the redistribution of organic material and consequently, the release of biochemical elements such as N, P, K and calcium and reduce the level of carbon content and C/N ratio of the soil. Our field microcosm study included population density and estimation biochemical compounds. Considering the data on the occurrence of millipedes, two sites were identified at study area as Site I (with millipede occurrence) and Site II (without millipedes). The soil samples from both sites were subjected for the analysis of biochemical compounds such as organic carbon, nitrogen, phosphorus, potassium and calcium. The site which is occupied by millipede (site I) showed remarkably high quantity of nitrogen (2.14%), phosphorus (23.10%), potassium (0.24%) and calcium (0.83%) than the site without millipedes (site II) during the month of November 2016. Similarly, an ideal C/N ratio (20.05) was observed in site I which favours the plant growth, in turn increases the quantity of leaf litter which supports the life of millipedes. Millipedes play an essential role in enhancing the fertility of soil and ecosystem functioning in this tropical dry deciduous forest of Alagar hill (Eastern India. In forest ecosystem, conservation of millipede is an essential step to improve the fertility of soil and maintaining the forest ecosystem.


INTRODUCTION
F orest floor varies in the quantity of plant debris and this variation is attributed to spatial heterogeneity of soil biota in forest ecosystem because plant debris provides them with food and shelter (David et al., 2006). Soil fauna are the important component in forest ecosystem, due to their functional role in the acceleration of organic matter, decomposition and nutrient transformation (Muneto et al., 2004;Bardgett et al., 2001). In the forest ecosystem, macro-arthropods ingest and accelerate the plant debris and nutritionally enriched the soil fertility (Nevo and Dror, 2021). The soil invertebrates have been shown to perform a significant character in plant litter decomposition in tropical forest (Liu and Zou, 2002). Soil macro creatures make significant commitments to the reusing of dead plant material across the globe (Griffiths et al., 2020). Decomposition process of plant residues is also influenced by substrate quality, decomposer community and environmental factors (Smith and Bradford, 2003). Shaw and Harte (2001) reported that litter decay and nutrient release are controlled by the litter quality, including the nitrogen (N) concentration of the litter, the carbon to nitrogen (C/N) ratio as well as other chemical properties.
Plant litter decomposition rates are often connected with lower C/N ratios and high nitrogen concentration (Mekonnen, 2020). On the other hand, soil macro-fauna gives a vital contribution to soil fertility by promoting the stability and productivity of forest ecosystem, mainly due to their influence on soil processes such as litter decomposition and nutrient dynamics (Nevo and Dror, 2021;Griffiths et al., 2020). According to Bird et al. (2000), established arthropod communities stabilize the availability of nutrients to plants and change the physical conditions of the soil, which become more suitable for root growth and plant water uptake. These conditions are important for maintaining productivity. Wolters (2000) reported that millipedes, woodlice, earthworms and slugs are the major soil macro fauna demonstrated to increase the nutrient leaching from dead-leaf litter and enhance the humification of the soil. Millipedes and earthworms are called "litter transformers and ecosystem engineers" O n l i n e F i r s t A r t i c l e (Kaneko and Ito, 2004). Moreover, the significant character of millipedes in decomposition is to alter the physical and chemical atmosphere of the litter. Millipedes nourish by shredding leaf litter with tooth-like structures on paired mandibles, and then crushing the shredded material between smooth molar plates (Sridhar and Ashwini, 2016). The nutrient content of the consumed plant resources also alters in the route through the millipede gut; pH, moisture and bacterial fungal counts are higher in faecal pellets than in the ingested litter (Tajovsky and Wytwe, 2009). In forest ecosystem, the pill millipede species, Glomeris marginata increase the microbial diversity simultaneously stimulating the nitrogen mineralization and enlistment of cations such as potassium and sodium (Andrew et al., 2006). Besides, feacal pellet formation results in an increase the availability of nutrients such as nitrogen and phosphorous to microorganisms (Scheu et al., 2005). Millipedes as detritivores, apparently affect nutrient cycling through the redistribution of organic material and, consequently, the release of chemical elements such as nitrogen in the soil (Sridhar and Ashwini, 2016). Since nutrient cycling is one of the key processes governing ecosystem dynamics (Tilman et al., 2014), millipedes may have profound influences on ecosystem. However, in forest habitats the millipede communities are influenced by the type of humus (Meyer and Singer, 1997), nitrogen (Stasiov, 2005) and carbon content (Stasiov, 2002), carbon-nitrogen ratio (Branquart et al., 1995), and pH of the soil (Smith et al., 2006). Even though, millipedes are the significant fauna in forest ecosystem, so far no adequate information is available on role of millipedes in soil fertility of forest ecosystem in India. Therefore, the major objective of the present study is to investigate the role of millipedes in soil fertility of Alagar Hill Reserve Forest, Madurai, Tamil Nadu.

MATERIALS AND METHODS
Observation of field microcosm studies were conducted at Silambar valley of Alagar Hills Reserve Forest from June 2016 to May 2018. The study area, Algarmalai Hills is located in Madurai District, a part of the eastern plains of the Deccan plateau, has many minor discontinuous hill ranges and isolated iselbergs of the Eastern Ghats. The area surveyed (10 o 0'-10 o 30'and 75 o 55'-78 o 20'E) are 20 km north-east of Madurai city. Two sites were identified at a stretch of 100 m distance long and 100 m transect i.e. Site I (with millipede occurrence) and Site II (without millipedes). Soil samples were collected from 5 quadrats each with 1m × 1m size from site I and II. Soil with litter was excavated up to 10cm depth from each sampling point twice a month from June 2016 to May 2018. For each study, different stretch of transects were used for assessment to avoid repeated sampling in the same site. The juvenile and adults of five millipede species, Harpaphe haydeniana, Xenobolus carnifex, Arthrosphaera magna, Aulacobolus newtoni and Spinotarsus colosseus were sampled in surface leaf litter.
The average number of individuals in the four quadrates of the study area was recorded. The mean of four such censuses collected in each month was considered the mean density of the population and expressed as number of individuals/m 2 . The soil samples were brought to the laboratory in airtight polythene bags for analysis of biochemical composition of soil. Composite sample of soil was mixed with distilled water (1:2.5w/v) and then shaken for 10 minutes. The pH was measured on the sampling spot using potable pH Meter (Systronics model 335, India). To quantify the organic Carbon in the compost, Walkley and Black's rapid titration method was followed (Jackson, 1973). Total nitrogen was determined using MicroKjeldhal method (Umbreit et al., 1974). Phosphorus was estimated by Vogel's technique (1963). Available potassium and calcium were detected using Flame Photometer (Elico; Model CL 378).

RESULTS
During the study period, maximum (34.2 o C) and minimum (21.5 o C) temperature were recorded in the month of May, 2017 and November, 2017, respectively. Whereas maximum (93%) and minimum (58%) humidity was recorded in November 2017 and May 2018, respectively. The highest rainfall (818.01mm) was observed in October 2017. The occurrence of millipede species was highest in November and it was found to decrease in the month of March to July. Density of five different millipede species was greater during 2016 -2017 than 2017 -2018 ( Fig.  1). The meteorological parameters such as temperature and humidity significantly altered population density and biomass of millipedes. A critical comparison of rainfall with the data on population density and biomass did not provide any significant conclusion.
Data related to pH, organic carbon, nitrogen, phosphorus, potassium, calcium and C/N ratio in the study site I and II during the period June 2016 to May 2018 are presented in Tables I to II. The pH value is comparatively much higher in study site II (6.3 to 7.1) than the site I (5.4 to 6.3) from June 2016 to May 2018. In study site I, the percentage of organic carbon was greater (50.02%) in the month of July 2017 and was much less (  The phosphorus content was maximum (23.10%) in the month of November 2016. But it was found to be less (4.89%) in the month of May 2018 in millipede occupying site (site I). In the site without millipedes (site II) phosphorus was found to be maximum (15.35%) in November 2017 and minimum (3.05%) in May 2018. The site I showed maximum amount of potassium content (0.24%) during the month of November 2016. But it was less (0.05%) in May 2017. In site II, maximum quantity of potassium (0.14%) was noticed in November 2016 and it was minimum (0.03%) in May 2018. With reference to calcium in study site I maximum (0.83%) and minimum (0.15%) values were obtained during the month of November 2016 and May 2018 respectively. In site II it was found to be maximum (0.45%) in November 2016 and minimum (0.08%) in May 2018. Two-way ANOVA indicates significant variations (P < 0.05) in sites with and without millipedes of Alagar hills reserve forest.

DISCUSSION
The soil fauna is a key component of forest ecosystem. They decompose the leaf litter and release the nutrient in the form of feacal pellet to the forest soil. The variation in the abundance for certain soil faunal groups and faunal diversity is often related to litter quantity and quality changes during succession and forest type (Wardle et al., 2006;Milcu et al., 2006). Diplopoda, the millipedes are one of the prominent soil fauna in forests. Their impact on decomposition processes and consequently soil element concentration may vary depending on the species. The quality of leaf litter in tropical region, influences the millipede population and, in turn, the rate of litter mineralization (Loranger et al., 2003(Loranger et al., , 2008. The feeding activity of the millipedes is also important in the vertical redistribution of organic matter in an ecosystem. As the animal feeds and travels vertically through the soil system, the aeration of the soil influences the flow of nutrients and propagation of microbes (David et al., 2001). Additionally, the fine particulate of the now comminuted litter has greater ability to move into the lower soil horizon by gravity. The combination of these modifications to the soil surface and litter allows for increased transfer on nutrients into the lower layers of the soil horizon through the flow of leachates. Smith et al. (2006) reported that, the pH values of the soil are the most important factors affecting the population density of millipedes in the urban parks and gardens of London. Influence of soil acidity on the communities of the millipedes was studied by Gonzalez et al. (2012) Soil fauna can enhance N concentration in litter by influencing N release and stimulation of microbial N mineralization (Gonzalez and Seastedt, 2001;Irmler, 2000). Similarly, the significantly higher concentration of N in soil subjected to millipede activity suggests that millipedes may play an important role in the mobilization of N, especially considering that N is a restrictive element in maximum ecosystems (Hooper et al., 2005). Warren and Zou (2002) observed a significant positive correlation between the density of millipedes and the nitrogen content of Leucaena plantation of Puerto Rico. Parafontaria laminata increased nitrogen mineralization with discharge excretion as soil aggregates (Okai et al., 2008). Loranger et al. (2008) has O n l i n e also demonstrated high feeding preference of nitrogenrich leaf litter by three millipede species in semi-evergreen tropical forest of Guadeloupe. Krishna and Mohan (2017) showed that soil fauna significantly increased the release of N in microcosms that enriched coniferous forest floors. Several workers have demonstrated the role of micro and macro arthropods in regulating C/N ratio during litter decomposition and humification processes. Yang and Chen (2009) stated that the soil fauna decrease carbon concentration and enhance nitrogen concentration in the rain forest leaf litter, where the initial high C/N ratio is substantially different from initial conditions in the broad leaf forest. Diplopoda (millipedes) as detritivores and macro fauna have been experimentally shown to have a significant effect on the mass loss of high C/N ratio of litter comparing to low C/N ratio litter (Hattenschwiler and Gasser, 2005). For example, influence of high density of millipede on litter decomposition of mixed litter with high C/N ratio in forest site is seen in increasing N concentration and decreasing C/N ratio (Irmler, 2000;Warren and Zou, 2002). Teuben and Verhoef (1992) reported that, the processing of litter by millipedes decreases the carbonnitrogen ratio in the fecal pellets. This is mostly caused by the increase in nitrogen compounds with relatively little change taking place in the amount of Ca. From the present study it is evident that the drastic reduction of C/N ratio in site I may be due to the occurrence of millipedes and that favored the growth of plants.

F i r s t A r t i c l e
The significant increase in K concentrations in the soil collected from site I may be due to high amount of faeces egested by millipedes that could increase the availability of K as reported by (Teuben and Verhoef, 1992). Ulrich (2000) O n l i n e

F i r s t A r t i c l e
Millipede Species Enrich the Fertility of Soil noted that level of sodium and potassium concentration increased rapidly and easily in leaf litter due to the impact of faunal activity. Consumption of litter by soil saprophagous fauna replenishes the soil nutrients and prevents elemental leaching by rain (Gonzalez and Seastedt, 2001). Diplopod is important sinks for calcium and accumulates Ca and Mg up to fivefold higher than raw leaf litter (Gonzalez and Seastedt, 2001). The faecal pellets of millipede remain stable for long duration without any changes (Ambarish and Sridhar, 2013). Such stability has been connected to high Ca ions in soil (Sridhar and Kadamannaya, 2009). Furthermore, Kaneko (1999) found that digestion of litter and soil by P. toniminea also amplified soil respiration and leaching of Ca + , Mg + . Ashwini and Sridhar (2006) detected a positive correlation in pill millipede richness and biomass to soil calcium content in Western Ghats. The presence of millipedes positively changes concentrations of many nutrients, not just ammonia, phosphorus, calcium and magnesium (Smit and van Aarde, 2001). Leaching of Ca, Mg and nitrogen increased in proportion to the number of millipedes. An increase in nitrate leaching can be explained by animal-feeding activity. Yang and Chen (2009) have reported the importance of preserving soil fauna diversity in forest for the process of nutrient cycling.
The present study indicates that the biochemical constituents such as nitrogen, phosphorus, potassium and Calcium are increased and favourable C/N ratio was observed in millipede occurrence site compared to millipede absent site of Alagar hills reserve forest. Fujimaki et al. (2010) found that the soil surface in the habitat area of P. laminate contained high amounts of O n l i n e

F i r s t A r t i c l e
R. Baluchamy and G. Radhakrishnan organic matter than in soil without the millipede because the faecal cast is higher in organic matter. In conclusion, this study indicated that millipede species assemblage provided a significant contribution to improve the soil fertility of Alagar hills reserve forest. Our present study corroborates earlier reports that the quality of leaf litter is important in enhancing the growth and food conversion efficiency of millipedes that, in turn, leads to leaf litter mineralization and soil enrichment through faecal egestion (Seeber et al., 2008). A similar study was observed by Topp et al. (2006) from millipedes collected from four primeval forests (including three oak-horbeam forest stands) of West Carpathians, Central Slovakia. They found that the gradient of the chemistry of the upper soil layer i.e. pH and nutrient content are strongly influenced by the millipede species. Similarly, the burrowing and other activities of millipede species can cause important physical modifications in the soil biochemical components in Algar Hill reserve forest. It is evident from the present study that the increase in biochemical constituents like, N, P, K, Ca during the month of November is because of increase in the quantity of millipede faeces during rainy seasons as also reported by Ashwini and Sridhar (2006). It has confirmed the findings of Dangerfied and Telford (1996) that leaf litter ingestion and faecal production by millipedes occur mainly during two month of wet season in South Africa. Millipede faeces consist of pulverized litter, minerals, and, high microbial load (Maraun et al., 2003). The significant amount of organ minerals in millipede fecal pellets accelerate humification of litter strata (Loranger et al., 2003). It has also been established that the faeces of Glomeris are a food source for certain earthworm species (Wolters, 2000). This proves that organominerals are re-ingested and mixed into the soil by earthworms, which cohabit at high biomasses with macroarthropods in mediterranean forests (Jordi et al., 2004). Soil macrofauna give an important contribution to soil fertility, by promoting the stability and productivity of forest ecosystem, mainly due to their influence on soil processes such as litter decomposition and nutrient dynamics (Irmler, 2000;Hunter et al., 2003;Griffiths et al., 2020). The present study emphasizes the importance of preserving millipede diversity in forest for the process of nutrient cycling and soil enrichment.

CONCLUSION
In forest ecosystem, soil macrofauna play an essential role in decomposition of litter, acceleration of organic matter and nutrient transformation. Among the litter arthropods, millipede is a significant fauna to alter the physical and chemical atmosphere of the litter. The site which is occupied by millipede contain remarkably high nitrogen, phosphorus, potassium and calcium. The minimum level of carbon observed in millipede presence site when compare the without millipede site which is make to ideal C/N ratio in forest ecosystem. Hence, the present study instructed that the preserving of millipede diversity in forest ecosystem is an essential step for the process of nutrient cycling and soil enrichment. It is significant to proliferate the awareness of ecologists about the importance of millipede diversity in forest ecosystem.