Resource partitioning among bat species in Peninsular Malaysia rice fields.

Resource partitioning among tropical bats in agricultural areas of Peninsular Malaysia remains unclear. This study was conducted to evaluate resource partitioning among bats by examining their fecal samples. The main bat species sampled included: Rhinolophus coelophyllus, Rhinolophus malayanus, Rhinolophus pusillus, Rhinolophus refulgens, Taphozous melanopogon and Hipposideros larvatus. Two harp traps were set at different elevations on a hilltop (Gunung Keriang) and two high nets were used in neighboring rice fields at three sites, for three consecutive nights per sampling from April 2021 to February 2022. A total of 301 bats and 1,505 pellets were analyzed using a conventional approach which examined the fecal sample under the microscope. All of the bat species within the study had insects from the order Coleoptera, Lepidoptera, Diptera and Hemiptera in their diet. Larger bats exhibited a greater variety of prey consumption. Male individuals were observed to be generalists while female individuals were specialists, particularly during pregnancy and lactating reproductive stages. Bat species and insect order had a significant impact on the percentage fragment frequency of the insects consumed. Rhinolophus coelophyllus specialized in feeding on Coleoptera and Diptera, H. larvatus fed on Coleoptera, R. malayanus fed on Hemiptera, R. pusillus and T. melanopogon fed on Lepidoptera. Future molecular analysis can be carried out to further identify the insect pests consumed by these bats up to species level. These findings enhance our understanding of bats' ecological roles in agricultural landscapes and contribute to conservation and pest management strategies.

Factors influencing the difference in dissolved ion inputs to the forest floor between deciduous and coniferous stands: comparison under high and low atmospheric deposition conditions.

It is necessary to clear the relationship between physical and vegetation factors on the processes governing dissolved ion inputs to the forest floor to estimate correctly the values of atmospheric input to the forest. This study identified the factors influencing the differences in dissolved ion inputs to the forest floor between coniferous evergreen and broad-leaved deciduous species by analyzing the phenological variations of dry deposition and canopy exchange calculated by the canopy budget model under a high-deposition site near the city of Tokyo and a low-deposition site 84 km further away. At low-deposition site, vegetation factors such as capture efficiency did not explain the differences in Na+ or Cl- dry deposition. Leaf physiological characteristics influenced the differences in the Mg2+ and Ca2+ canopy leaching values, and phenology, leaf wettability, and diffusion processes from water film into leaves influenced the differences in NH4+ and NO3- input processes between tree types. At the high-deposition site, differences in the dry deposition of Na+, SO42-, Cl-, Mg2+, Ca2+, NH4+, and NO3- between tree types were influenced by differences in capture efficiency between coniferous and broad-leaved canopies in the leafed period and by the absence of leaves in deciduous species after leaf fall. These results indicated that atmospheric deposition affected the capture efficiency of coniferous trees for dry deposition and enhanced the difference of dissolved ion inputs to the forest floor between coniferous and deciduous species.

Striped catfish (Pangasianodon hypophthalmus) exploit food sources across anaerobic decomposition- and primary photosynthetic production-based food chains.

Dietary information from aquatic organisms is instrumental in predicting biological interactions and understanding ecosystem functionality. In freshwater habitats, generalist fish species can access a diverse array of food sources from multiple food chains. These may include primary photosynthetic production and detritus derived from both oxic and anoxic decomposition. However, the exploitation of anoxic decomposition products by fish remains insufficiently explored. This study examines feeding habits of striped catfish (Pangasianodon hypophthalmus) at both adult and juvenile stages within a tropical reservoir, using stable carbon, nitrogen, and sulfur isotope ratios (δ13C, δ15N, and δ34S, respectively) and fatty acid (FA) analyses. The adult catfish exhibited higher δ15N values compared to primary consumers that feed on primary photosynthetic producers, which suggests ingestion of food sources originating from primary photosynthetic production-based food chains. On the other hand, juvenile catfish demonstrated lower δ15N values than primary consumers, correlating with low δ34S value and large proportions of bacterial FA but contained small proportions of polyunsaturated FA. This implies that juveniles utilize food sources from both anoxic decomposition and primary photosynthetic production-based food chains. Our results indicate that food chains based on anoxic decomposition can indeed contribute to the dietary sources of tropical fish species.

Effect of radioactive cesium-rich microparticles on radioactive cesium concentration and distribution coefficient in rivers flowing through the watersheds with different contaminated condition in Fukushima.

Radioactive cesium-rich microparticles (CsMPs) derived from the Fukushima Daiichi Nnuclear Power Plant accident were detected from soils and river water around Fukushima Prefecture, Japan. Because CsMPs are insoluble and rich in radioactive cesium (RCs), they may cause the overestimation of solid-water distribution coefficient (Kd) for RCs in the water. Previous studies showed the proportion of RCs derived from CsMPs on RCs concentration in soils collected from areas with different contaminated levels. Because the proportion of RCs concentration derived CsMPs to the RCs concentration of soils in the less contaminated areas is higher than that in the highly contaminated areas, the effect of CsMPs on particulate RCs concentration in river water may be larger in the less contaminated areas. However, the difference in the effects of CsMPs on the particulate RCs concentration and Kd in river water flowing through watersheds with different contaminated levels has not been clarified. In this study, we investigated the effect of CsMPs on the particulate RCs concentration and Kd in two rivers, Takase River and Kami-Oguni River, flowing through the watersheds with different RCs contaminated levels in Fukushima Prefecture. CsMPs might enter rivers due to soil erosion because they were detected only in some samples collected from both rivers during flood events. CsMPs accounted for more than half of particulate RCs concentration in some water samples collected in the flood condition. In particular, the proportion of CsMPs in particulate RCs for the Kami-Oguni River was greater than that for the Takase River. However, when evaluating for the entire water sampling in the flood condition, a proportion of RCs concentration derived from CsMPs in the average RCs concentrations per unit mass of SS in both river waters collected in the flood condition was not large. CsMPs might temporarily increase the particulate RCs concentration and Kd in the flood event, but CsMPs did not significantly affect them when evaluated throughout the event.

CESIUM-RICH MICROPARTICLES RUNOFF DURING RAINFALL: A CASE STUDY IN THE TAKASE RIVER.

Cesium-rich microparticles (CsMPs) with high cesium-137 (137Cs) concentrations were released and deposited in surface soil after the Fukushima Daiichi Nuclear Power Plant accident. Radioactive materials on the soil surface layer enter rivers owing to soil erosion during rainfall. In this study, we investigated CsMPs runoff through the river via soil erosion during rainfall in the Takase River watershed in Namie Town, Fukushima Prefecture, Japan. CsMPs were rarely detected in suspended solids (SS) in water samples collected during four rainfalls between February and July 2021. Furthermore, the proportion of 137Cs concentration derived from CsMPs to 137Cs concentration in the form of SS (particulate 137Cs) in the water was ~6% on average, which suggests that 137Cs runoff in the form of CsMPs from the forest to the Takase River was not large.

Roosting ecology of endangered plant-roosting bats on Okinawa Island: Implications for bat-friendly forestry practices.

Roosting information is crucial to guiding bat conservation and bat-friendly forestry practices. The Ryukyu tube-nosed bat Murina ryukyuana (Endangered) and Yanbaru whiskered bat Myotis yanbarensis (Critically Endangered) are forest-dwelling bats endemic to the central Ryukyu Archipelago, Japan. Despite their threatened status, little is known about the roosting ecology of these species and the characteristics of natural maternity roosts are unknown. To inform sustainable forestry practices and conservation management, we radio-tracked day roosts of both species in the subtropical forests of Okinawa's Kunigami Village District. We compared roost and roost site characteristics statistically between M. ryukyuana nonmaternity roosts (males or nonreproductive females), maternity roosts, and all M. yanbarensis roosts. Generalized linear models were used to investigate roost site selection by M. ryukyuana irrespective of sex and age class. Lastly, we compiled data on phenology from this and prior studies. Nonreproductive M. ryukyuana roosted alone and primarily in understory foliage. Murina ryukyuana maternity roosts were limited to stands >50 years old, and ~60% were in foliage. Myotis yanbarensis roosted almost entirely in cavities along gulch bottoms and only in stands >70 years old (~1/3 of Kunigami's total forest area). Murina ryukyuana maternity roosts were higher (4.3 ± 0.6 m) than conspecific nonmaternity roosts (2.3 ± 0.5 m; p < .001) and M. yanbarensis roosts (2.7 ± 0.5 m; not significant). Model results were inconclusive. Both species appear to be obligate plant roosters throughout their life cycle, but the less flexible roosting preferences of M. yanbarensis may explain its striking rarity. To conserve these threatened bats, we recommend the following forestry practices: (a) reduce clearing of understory vegetation, (b) refrain from removing trees along streams, (c) promote greater tree cavity densities by protecting old-growth forests and retaining snags, and (d) refrain from removing trees or understory between April and July, while bats are pupping.

Respective contribution of urban wastewater and mangroves on nutrient dynamics in a tropical estuary during the monsoon season.

Estuaries of Southeast Asia are increasingly impacted by land-cover changes and pollution. Here, our research objectives were to (1) determine the origins of nutrient loads along the Can Gio estuary (Vietnam) and (2) identify the processes that affect the nutrient pools during the monsoon. We constructed four 24-h time-series along the salinity gradient measuring nutrient concentrations and stable isotopes values. In the upper estuary, urban effluents from Ho Chi Minh City were the main input of nutrients, leading to dissolved oxygen saturation <20%. In the lower estuary, ammonium and nitrite concentration peaks were explained by mangrove export. No contribution from aquaculture was detected, as it represents <0.01% of the total river discharge. Along the salinity gradient, nutrient inputs were rapidly consumed, potentially by phytoplankton while nitrate dual-stable isotopes indicated that nitrification occurred. Thus, even in a large and productive estuary, urban wastewater can affect nutrient dynamics with potentially important ecological risks.

Dissimilatory Nitrate Reduction to Ammonium and Responsible Microbes in Japanese Rice Paddy Soil.

Nitrification-denitrification processes in the nitrogen cycle have been extensively examined in rice paddy soils. Nitrate is generally depleted in the reduced soil layer below the thin oxidized layer at the surface, and this may be attributed to high denitrification activity. In the present study, we investigated dissimilatory nitrate reduction to ammonium (DNRA), which competes with denitrification for nitrate, in order to challenge the conventional view of nitrogen cycling in paddy soils. We performed paddy soil microcosm experiments using 15N tracer analyses to assess DNRA and denitrification rates and conducted clone library analyses of transcripts of nitrite reductase genes (nrfA, nirS, and nirK) in order to identify the microbial populations carrying out these processes. The results obtained showed that DNRA occurred to a similar extent to denitrification and appeared to be enhanced by a nitrate limitation relative to organic carbon. We also demonstrated that different microbial taxa were responsible for these distinct processes. Based on these results and previous field observations, nitrate produced by nitrification within the surface oxidized layer may be reduced not only to gaseous N2 via denitrification, but also to NH4+ via DNRA, within the reduced layer. The present results also indicate that DNRA reduces N loss through denitrification and nitrate leaching and provides ammonium to rice roots in rice paddy fields.

Distinct Community Composition of Previously Uncharacterized Denitrifying Bacteria and Fungi across Different Land-Use Types.

Recent studies demonstrated that phylogenetically more diverse and abundant bacteria and fungi than previously considered are responsible for denitrification in terrestrial environments. We herein examined the effects of land-use types on the community composition of those denitrifying microbes based on their nitrite reductase gene (nirK and nirS) sequences. These genes can be phylogenetically grouped into several clusters. We used cluster-specific PCR primers to amplify nirK and nirS belonging to each cluster because the most widely used primers only amplify genes belonging to a single cluster. We found that the dominant taxa as well as overall community composition of denitrifying bacteria and fungi, regardless of the cluster they belonged to, differed according to the land-use type. We also identified distinguishing taxa based on individual land-use types, the distribution of which has not previously been characterized, such as denitrifying bacteria or fungi dominant in forest soils, Rhodanobacter having nirK, Penicillium having nirK, and Bradyrhizobium having nirS. These results suggest that land-use management affects the ecological constraints and consequences of denitrification in terrestrial environments through the assembly of distinct communities of denitrifiers.

Consequences of microbial diversity in forest nitrogen cycling: diverse ammonifiers and specialized ammonia oxidizers.

We tested the ecosystem functions of microbial diversity with a focus on ammonification (involving diverse microbial taxa) and nitrification (involving only specialized microbial taxa) in forest nitrogen cycling. This study was conducted on a forest slope, in which the soil environment and plant growth gradually changed. We measured the gross and net rates of ammonification and nitrification, the abundance of predicted ammonifiers and nitrifiers, and their community compositions in the soils. The abundance of predicted ammonifiers did not change along the soil environmental gradient, leading to no significant change in the gross ammonification rate. On the other hand,  the abundance of nitrifiers and the gross nitrification rate gradually changed. These accordingly determined the spatial distribution of net accumulation of ammonium and nitrate available to plants. The community composition of predicted ammonifiers gradually changed along the slope, implying that diverse ammonifiers were more likely to include taxa that were acclimated to the soil environment and performed ammonification at different slope locations than specialized nitrifiers. Our findings suggest that the abundance of ammonifiers and nitrifiers directly affects the corresponding nitrogen transformation rates, and that their diversity affects the stability of the rates against environmental changes. This study highlights the role of microbial diversity in biogeochemical processes under changing environments and plant growth.

Unprocessed Atmospheric Nitrate in Waters of the Northern Forest Region in the U.S. and Canada.

Little is known about the regional extent and variability of nitrate from atmospheric deposition that is transported to streams without biological processing in forests. We measured water chemistry and isotopic tracers (δ18O and δ15N) of nitrate sources across the Northern Forest Region of the U.S. and Canada and reanalyzed data from other studies to determine when, where, and how unprocessed atmospheric nitrate was transported in catchments. These inputs were more widespread and numerous than commonly recognized, but with high spatial and temporal variability. Only 6 of 32 streams had high fractions (>20%) of unprocessed atmospheric nitrate during baseflow. Seventeen had high fractions during stormflow or snowmelt, which corresponded to large fractions in near-surface soil waters or groundwaters, but not deep groundwater. The remaining 10 streams occasionally had some (<20%) unprocessed atmospheric nitrate during stormflow or baseflow. Large, sporadic events may continue to be cryptic due to atmospheric deposition variation among storms and a near complete lack of monitoring for these events. A general lack of observance may bias perceptions of occurrence; sustained monitoring of chronic nitrogen pollution effects on forests with nitrate source apportionments may offer insights needed to advance the science as well as assess regulatory and management schemes.

Using food network unfolding to evaluate food-web complexity in terms of biodiversity: theory and applications.

Food-web complexity often hinders disentangling functionally relevant aspects of food-web structure and its relationships to biodiversity. Here, we present a theoretical framework to evaluate food-web complexity in terms of biodiversity. Food network unfolding is a theoretical method to transform a complex food web into a linear food chain based on ecosystem processes. Based on this method, we can define three biodiversity indices, horizontal diversity (DH ), vertical diversity (DV ) and range diversity (DR ), which are associated with the species diversity within each trophic level, diversity of trophic levels, and diversity in resource use, respectively. These indices are related to Shannon's diversity index (H'), where H' = DH  + DV  - DR . Application of the framework to three riverine macroinvertebrate communities revealed that D indices, calculated from biomass and stable isotope features, captured well the anthropogenic, seasonal, or other within-site changes in food-web structures that could not be captured with H' alone.

Effects of litter feeders on the transfer of 137Cs to plants.

The effects of the Japanese horned beetle larvae on the transfer of 137Cs from a contaminated leaf litter to the leaf vegetable, komatsuna (Brassica rapa var. perviridis) was studied. Feces of the larvae which were fed 137Cs-contaminated leaf litter were added to a potting mix in which komatsuna plants were cultivated. The presence of feces increased the harvest yield of komatsuna, suggesting that feces provided nutrients for the plant growth. In addition, the amount of exchangeable 137Cs in leaf litter was experimentally confirmed to be enhanced by the presence of feces which were excreted by larvae feeding. However, there was no difference in the soil-to-plant transfer factor of 137Cs for the presence and absence of feces. Interactions between clay minerals and exchangeable 137Cs in the soil beneath the litter layer may diminish the root uptake of 137Cs. From these results, it was concluded that the effect of exchangeable 137Cs released from feces was limited for the transfer of 137Cs to plants if plant roots were not present in litter layers.

Spatial variations in the molecular diversity of dissolved organic matter in water moving through a boreal forest in eastern Finland.

Dissolved organic matter (DOM) strongly affects water quality within boreal forest ecosystems. However, how the quality of DOM itself changes spatially is not well understood. In this study, to examine how the diversity of DOM molecules varies in water moving through a boreal forest, the number of DOM molecules in different water samples, i.e., rainwater, throughfall, soil water, groundwater, and stream water was determined using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in Norway spruce and Scots pine stands in eastern Finland during May and June 2010. The number of molecular compounds identified by FT-ICR MS (molecular diversity) ranged from 865 to 2,194, revealing large DOM molecular diversity in the water samples. Additionally, some of the molecular compounds were shared between different water samples. The DOM molecular diversity linearly correlated with the number of low-biodegradable molecules, such as, lignin-like molecules (lignins), but not with dissolved organic carbon concentration. The number of lignins shared between different sampling locations was larger than that of any other biomolecular class. Our results suggest that low-biodegradable molecules, especially lignins, regulate spatial variations in DOM molecular diversity in boreal forests.

Terrestrial-aquatic linkage in stream food webs along a forest chronosequence: multi-isotopic evidence.

Long-term monitoring of ecosystem succession provides baseline data for conservation and management, as well as for understanding the dynamics of underlying biogeochemical processes. We examined the effects of deforestation and subsequent afforestation of a riparian forest of Japanese cedar (Cryptomeria japonica) on stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹5N) and natural abundances of radiocarbon (Δ¹4C) in stream biota in the Mt. Gomadan Experimental Forest and the Wakayama Forest Research Station, Kyoto University, central Japan. Macroinvertebrates, periphytic algae attached to rock surfaces (periphyton), and leaf litter of terrestrial plants were collected from six headwater streams with similar climate, topography, and bedrock geology, except for the stand ages of riparian forests (from 3 to 49 yr old in five stands and > 90 yr old in one reference stand). Light intensity and δ¹³C values of both periphyton and macroinvertebrates decreased synchronously with forest age in winter. A Bayesian mixing model indicates that periphyton contributions to the stream food webs are maximized in 23-yr-old forests. Except for grazers, most macroinvertebrates showed Δ¹4C values similar to those of terrestrial leaf litter, reflecting the influence of modern atmospheric CO2 Δ¹4C values. On the other hand, the Δ¹4C values of both periphyton and grazers (i.e., aquatic primary consumers) were significantly lower than that of modern atmospheric CO2, and were lowest in 23-yr-old forest stands. Previous studies show that root biomass of C. japonica peaks at 15-30 yr after planting. These evidences suggest that soil CO2 released by root respiration and dispersed by groundwater weathers carbonate substrata, and that dissolved inorganic carbon (DIC) with low Δ¹4C is incorporated into stream periphyton and some macroinvertebrates. The ecological response in the studied streams to clear-cutting and replanting of Japanese cedar is much slower (~20 yr) than the chemical response (< 5 yr). More than 50 yr is required for the food web structure to completely recover from clear-cutting. The ecological delay is attributed to several biogeochemical factors, the understanding of which is critical to integrated management of forest-stream continuum and the prediction of ecosystem resilience in response to environmental change.

(137)Cs distributions in soil and trees in forest ecosystems after the radioactive fallout - Comparison study between southern Finland and Fukushima, Japan.

The nuclear accidents at Chernobyl and Fukushima released large amounts of (137)Cs radionuclides into the atmosphere which spread over large forest areas. We compared the (137)Cs concentration distribution in different parts of two coniferous forest ecosystems (needle litter, stems and at different depths in the soil) over short and long term periods in Finland and Japan. We also estimated the change in (137)Cs activity concentrations in needle and soil between 1995 and 2013 in Southern Finland based on the back-calculated (137)Cs activity concentrations. We hypothesized that if the (137)Cs activity concentrations measured in 1995 and 2013 showed a similar decline in concentration, the (137)Cs activity concentration in the ecosystem was already stable in 1995. But if not, the (137)Cs activity concentrations were still changing in 2013. Our results showed that the vertical distribution of the (137)Cs fallout in the soil was similar in Hyytiälä and Fukushima. The highest (137)Cs concentrations were observed in the uppermost surface layers of the soil, and they decreased exponentially deeper in the soil. We also observed that (137)Cs activity concentrations estimated from the samples in 1995 and 2013 in Finland showed different behavior in the surface soil layers compared to the deep soil layer. These results suggested that the (137)Cs nuclei were still mobile in the surface soil layers 27 years after the accident. Our results further indicated that, in the aboveground parts of the trees, the (137)Cs concentrations were much closer to steady-state when compared to those of the surface soil layers based on the estimated declining rates of (137)Cs concentration activity in needles which were similar in 1995 and 2013. Despite its mobility and active role in the metabolism of trees, the (137)Cs remains in the structure of the trees for decades, and there is not much exchange of (137)Cs between the heartwood and surface layers of the stem.

Role of natural organic matter on iodine and (239)(,240)Pu distribution and mobility in environmental samples from the northwestern Fukushima Prefecture, Japan.

In order to assess how environmental factors are affecting the distribution and migration of radioiodine and plutonium that were emitted from the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, we quantified iodine and (239,240)Pu concentration changes in soil samples with different land uses (urban, paddy, deciduous forest and coniferous forest), as well as iodine speciation in surface water and rainwater. Sampling locations were 53-63 km northwest of the FDNPP within a 75-km radius, in close proximity of each other. A ranking of the land uses by their surface soil (<4 cm) stable (127)I concentrations was coniferous forest > deciduous forest > urban > paddy, and (239,240)Pu concentrations ranked as deciduous forest > coniferous forest > paddy ≥ urban. Both were quite distinct from that of (134)Cs and (137)Cs: urban > coniferous forest > deciduous forest > paddy, indicating differences in their sources, deposition phases, and biogeochemical behavior in these soil systems. Although stable (127)I might not have fully equilibrated with Fukushima-derived (129)I, it likely still works as a proxy for the long-term fate of (129)I. Surficial soil (127)I content was well correlated to soil organic matter (SOM) content, regardless of land use type, suggesting that SOM might be an important factor affecting iodine biogeochemistry. Other soil chemical properties, such as Eh and pH, had strong correlations to soil (127)I content, but only within a given land use (e.g., within urban soils). Organic carbon (OC) concentrations and Eh were positively, and pH was negatively correlated to (127)I concentrations in surface water and rain samples. It is also noticeable that (127)I in the wet deposition was concentrated in both the deciduous and coniferous forest throughfall and stemfall water, respectively, comparing to the bulk rainwater. Further, both forest throughfall and stemflow water consisted exclusively of organo-iodine, suggesting all inorganic iodine in the original bulk deposition (∼ 28.6% of total iodine) have been completely converted to organo-iodine. Fukushima-derived (239,240)Pu was detectable at a distance ∼ 61 km away, NW of FDNPP. However, it is confined to the litter layer, even three years after the FDNPP accident-derived emissions. Plutonium-239,240 activities were significantly correlated with soil OC and nitrogen contents, indicating Pu may be associated with nitrogen-containing SOM, similar to what has been observed at other locations in the United States. Together, these finding suggest that natural organic matter (NOM) plays a key role in affecting the fate and transport of I and Pu and may warrant greater consideration for predicting long-term stewardship of contaminated areas and evaluating various remediation options in Japan.

Estimation of radioactive 137-cesium transportation by litterfall, stemflow and throughfall in the forests of Fukushima.

Since the Fukushima Dai-ichi Nuclear Power Plant accident in March 2011, large areas of the forests around Fukushima have become highly contaminated by radioactive nuclides. To predict the future dynamics of radioactive cesium ((137)Cs) in the forest catchment, it is important to measure each component of its movement within the forest. Two years after the accident, we estimated the annual transportation of (137)Cs from the forest canopy to the floor by litterfall, throughfall and stemflow. Seasonal variations in (137)Cs transportation and differences between forests types were also determined. The total amount of (137)Cs transported from the canopy to the floor in two deciduous and cedar plantation forests ranged between 3.9 and 11.0 kBq m(-2) year(-1). We also observed that (137)Cs transportation with litterfall increased in the defoliation period, simply because of the increased amount of litterfall. (137)Cs transportation with throughfall and stemflow increased in the rainy season, and (137)Cs flux by litterfall was higher in cedar plantation compared with that of mixed deciduous forest, while the opposite result was obtained for stemflow.

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously.

Denitrification is an important process in the global nitrogen cycle. The genes encoding NirK and NirS (nirK and nirS), which catalyze the reduction of nitrite to nitric oxide, have been used as marker genes to study the ecological behavior of denitrifiers in environments. However, conventional polymerase chain reaction (PCR) primers can only detect a limited range of the phylogenetically diverse nirK and nirS. Thus, we developed new PCR primers covering the diverse nirK and nirS. Clone library and qPCR analysis using the primers showed that nirK and nirS in terrestrial environments are more phylogenetically diverse and 2-6 times more abundant than those revealed with the conventional primers. RNA- and culture-based analyses using a cropland soil also suggested that microorganisms with previously unconsidered nirK or nirS are responsible for denitrification in the soil. PCR techniques still have a greater capacity for the deep analysis of target genes than PCR-independent methods including metagenome analysis, although efforts are needed to minimize the PCR biases. The methodology and the insights obtained here should allow us to achieve a more precise understanding of the ecological behavior of denitrifiers and facilitate more precise estimate of denitrification in environments.