No evidence of carbon storage usage for seed production in 18 dipterocarp masting species in a tropical rain forest.

Most canopy species in lowland tropical rain forests in Southeast Asia, represented by Dipterocarpaceae, undergo mast reproduction synchronously at community level during a general flowering event. Such events occur at irregular intervals of 2-10 years. Some species do not necessarily participate in every synchronous mast reproduction, however. This may be due to a lack of carbohydrate resources in the trees for masting. We tested the hypothesis that interspecific differences in the time required to store assimilates in trees for seed production are due to the frequency of masting and/or seed size in each species. We examined the relationship between reproductive frequency and the carbon accumulation period necessary for seed production, and between the seed size and the period, using radiocarbon analysis in 18 dipterocarp canopy species. The mean carbon accumulation period was 0.84 years before seed maturation in all species studied. The carbon accumulation period did not have any significant correlation with reproductive frequency or seed size, both of which varied widely across the species studied. Our results show that for seed production, dipterocarp masting species do not use carbon assimilates stored for a period between the masting years, but instead use recent photosynthates produced primarily in a masting year, regardless of the masting interval or seed size of each species. These findings suggest that storage of carbohydrate resources is not a limiting factor in the masting of dipterocarps, and that accumulation and allocation of other resources is important as a precondition for participation in general flowering.

Partial mycoheterotrophy in the leafless orchid Eulophia zollingeri specialized on wood-decaying fungi.

Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies had shown that E. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid's entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstrated that E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi.

Sample pretreatment effects on isotopic compositions of oxygen and sulfur in BaSO4 derived from atmospheric sulfate.

RATIONALE: Preparation of BaSO4 from samples of atmospheric rain, snow, and aerosols has been used for δ18 O and δ34 S analyses. In the present study, we investigated the effect of various sample pretreatments on δ18 O and δ34 S values determined from a Na2 SO4 reagent solution and samples of atmospheric precipitation to improve assay time and cost efficiency. METHODS: BaSO4 was prepared from a Na2 SO4 solution by (a) evaporative concentration, (b) evaporation to dryness, (c) evaporation to dryness after adding HCl, and (d) evaporation to concentration after adding HCl, followed by cooling and then precipitation using a BaCl2 solution. To analyze the atmospheric precipitation samples for δ18 O, BaSO4 prepared from the samples was treated with diethylenetriaminepentaacetic acid (DTPA) and SO4 2- and separated chromatographically. The values of δ18 O and δ34 S were measured using a continuous-flow elemental analyzer coupled to an isotope-ratio mass spectrometer. RESULTS: The δ34 S values in BaSO4 precipitated from Na2 SO4 solution determined by methods (a)-(c) were consistent within precisions of ±0.5‰. The δ18 O values of methods (a) and (b) were consistent within ±0.2‰, whereas the δ18 O values of methods (c) and (d) increased with increasing HCl concentrations. Similar results were obtained from samples of atmospheric precipitations. The δ18 O values from DTPA-treated BaSO4 were consistent with those obtained by chromatographic separation within ±0.5‰. CONCLUSIONS: We found no significant differences in the effects of various pretreatments (acidification, heating, concentration, and drying) on δ18 O and δ34 S values in sulfate from samples of atmospheric precipitation and aerosols extracted as BaSO4 if HCl was not added to the sample before heating and BaSO4 was treated with DTPA for the δ18 O analysis.

Novel mycorrhizal cheating in a green orchid: Cremastra appendiculata depends on carbon from deadwood through fungal associations.

To date, there has been no robust evidence for the exploitation of saprotrophic non-rhizoctonia fungi by green plants, although some fully mycoheterotrophic orchids are known to exploit them, and mycoheterotrophic evolution has probably occurred through intermediate mixotrophic stages. We investigated the physiological ecology of a fully mycoheterotrophic species Cremastra aphylla and its photosynthetic sister species Cremastra appendiculata, which putatively exploit saprotrophic fungi. Their mycorrhizal partners and ultimate nutritional sources were determined using molecular, stable isotopic, and radiocarbon analysis. Both Cremastra aphylla and Cremastra appendiculata were consistently associated with wood-decaying Psathyrellaceae. In addition, both species were highly enriched in carbon-13 (13 C) and, to a less degree, in nitrogen-15 (15 N). The δ13 C and δ15 N values of Cremastra appendiculata were intermediate between those of Cremastra aphylla and those of autotrophic plants. All Cremastra appendiculata samples and two Cremastra aphylla samples exhibited elevated Δ14 C values due to the acquisition of carbon fixed in wood during the past decades (14 C-enriched bomb carbon). Our multifaceted evidence indicated that both species obtained carbon from deadwood via saprotrophic fungi. Our findings strongly suggest that mixotrophic relationships associated with wood-decaying fungi represent a novel evolutionary pathway for full mycoheterotrophy in orchids.

A new sampling method with zirconium-loaded resin for phosphate oxygen isotope analysis in oligotrophic freshwater systems.

RATIONALE: The phosphate oxygen isotope ratio ( δ 18 O PO 4 ) is a useful technique to trace the sources and biogeochemical cycles of phosphorus (P) in aquatic ecosystems. However, δ 18 O PO 4 has not been widely used in oligotrophic freshwater systems due to technical and methodological difficulties in collecting sufficient phosphate (PO4 ) for the δ 18 O PO 4 analysis, which sometimes requires hundreds of liters of the water sample. In this study, a new approach (PaS-Zir) was developed for the δ 18 O PO 4 analysis in oligotrophic freshwater systems using zirconium (Zr)-loaded (ZrIRC) resin, which has a high affinity for PO4 . METHODS: ZrClO2 was added to Amberlite IRC748 to obtain the ZrIRC resin. The adsorption/desorption experiment using KH2 PO4 with a known value of δ 18 O PO 4 was conducted to determine the adsorption/desorption properties of the resin and the likelihood of isotopic fractionation. By installing mesh bags filled with the resin, the PaS-Zir approach was used in two rivers with low PO4 concentrations (0.2 and 5.3 µmol/L). A conventional sampling method was also performed in the study river with a higher PO4 concentration to validate the efficacy of the PaS-Zir method. RESULTS: The adsorption/desorption experiment demonstrated that the ZrIRC resin possessed a sufficient adsorption capacity (153 µmol/resin-mL) and exhibited little isotopic fractionation during the adsorption/desorption processes. Using the PaS-Zir method, we were able to collect sufficient PO4 samples for the δ 18 O PO 4 analysis from the rivers within at least 4 days of mesh bag installation. The δ 18 O PO 4 values (14.2‰ ± 0.2‰) obtained using the PaS-Zir method were comparable to those obtained using the conventional method (14.0‰ ± 0.03‰). CONCLUSION: We proved that the PaS-Zir method is applicable to oligotrophic freshwater systems and is generally more efficient than the conventional method. In addition, our method is useful for improving the understanding of the P dynamics of oligotrophic ecosystems because of the extremely low concentration of PO4 commonly found in them, which are often prone to P pollution.

A new method for phosphate purification for oxygen isotope ratio analysis in freshwater and soil extracts using solid-phase extraction with zirconium-loaded resin.

RATIONALE: Phosphate (PO4 ) oxygen isotope (δ18 OPO4 ) analysis is increasingly applied to elucidate phosphorus cycling. Due to its usefulness, analytical methods continue to be developed and improved to increase processing efficiency and applicability to various sample types. A new pretreatment procedure to obtain clean Ag3 PO4 using solid-phase extraction (SPE) with zirconium-loaded resin (ZrME), which can selectively adsorb PO4 , is presented and evaluated here. METHODS: Our method comprises (1) PO4 concentration, (2) PO4 separation by SPE, (3) cation removal, (4) Cl- removal, and (5) formation of Ag3 PO4 . The method was tested by comparing the resulting δ18 OPO4 of KH2 PO4 reagent, soil extracts (NaHCO3 , NaOH, and HCl), freshwater, and seawater with data obtained using a conventional pretreatment method. RESULTS: PO4 recovery of our method ranged from 79.2% to 97.8% for KH2 PO4 , soil extracts, and freshwater. Although the recovery rate indicated incomplete desorption of PO4 from the ZrME columns, our method produced high-purity Ag3 PO4 and accurate δ18 OPO4 values (i.e., consistent with those obtained using conventional pretreatment methods). However, for seawater, the PO4 recovery was low (1.1%), probably due to the high concentrations of F- and SO4 2- which interfere with PO4 adsorption on the columns. Experiments indicate that the ZrME columns could be regenerated and used repeatedly at least three times. CONCLUSIONS: We demonstrated the utility of ZrME for purification of PO4 from freshwater and soil extracts for δ18 OPO4 analysis. Multiple samples could be processed in three days using this method, increasing sample throughput and potentially facilitating more widespread use of δ18 OPO4 analysis to deepen our understanding of phosphorus cycling in natural environments.

Radiocarbon signature reveals that most springtails depend on carbon from living plants.

Terrestrial carbon cycling is largely mediated by soil food webs. Identifying the carbon source for soil animals has been desired to distinguish their roles in carbon cycling, but it is challenging for small invertebrates at low trophic levels because of methodological limitations. Here, we combined radiocarbon (14C) analysis with stable isotope analyses (13C and 15N) to understand feeding habits of soil microarthropods, especially focusing on springtail (Collembola). Most Collembola species exhibited lower Δ14C values than litter regardless of their δ13C and δ15N signatures, indicating their dependence on young carbon. In contrast with general patterns across all taxonomic groups, we found a significant negative correlation between δ15N and Δ14C values among the edaphic Collembola. This means that the species with higher δ15N values depend on C from more recent photosynthate, which suggests that soil-dwelling species generally feed on mycorrhizae to obtain root-derived C. Many predatory taxa exhibited higher Δ14C values than Collembola but lower than litter, indicating non-negligible effects of collembolan feeding habits on the soil food web. Our study demonstrated the usefulness of radiocarbon analysis, which can untangle the confounding factors that change collembolan δ15N values, clarify animal feeding habits and define the roles of organisms in soil food webs.

Partial and full mycoheterotrophy in green and albino phenotypes of the slipper orchid Cypripedium debile.

Most green orchids form mycorrhizal associations with rhizoctonia fungi, a polyphyletic group including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae. Although accumulating evidence indicated that partial mycoheterotrophy occurs in such so-called rhizoctonia-associated orchids, it remains unclear how much nutrition rhizoctonia-associated orchids obtain via mycoheterotrophic relationships. We investigated the physiological ecology of green and albino individuals of a rhizoctonia-associated orchid Cypripedium debile, by using molecular barcoding of the mycobionts and stable isotope (13C and 15 N) analysis. Molecular barcoding of the mycobionts indicated that the green and albino individuals harbored Tulasnella spp., which formed a clade with the previously reported C. debile mycobionts. In addition, stable isotope analysis showed that both phenotypes were significantly enriched in 13C but not in 15 N. Therefore, green and albino individuals were recognized as partial and full mycoheterotrophs, respectively. The green variants were estimated to obtain 42.5 ± 8.2% of their C from fungal sources, using the 13C enrichment factor of albino individuals as a mycoheterotrophic endpoint. The proportion of fungal-derived C in green C. debile was higher than that reported in other rhizoctonia-associated orchids. The high fungal dependence may facilitate the emergence of albino mutants. Our study provides the first evidence of partial mycoheterotrophy in the subfamily Cypripedioideae. Partial mycoheterotrophy may be more general than previously recognized in the family Orchidaceae.

Specialized mycorrhizal association between a partially mycoheterotrophic orchid Oreorchis indica and a Tomentella taxon.

The evolution of full mycoheterotrophy in orchids likely occurs through intermediate stages (i.e., partial mycoheterotrophy or mixotrophy), in which adult plants obtain nutrition through both autotrophy and mycoheterotrophy. However, because of its cryptic manifestation, partial mycoheterotrophy has only been confirmed in slightly more than 20 orchid species. Here, we hypothesized that Oreorchis indica is partially mycoheterotrophic, since (i) Oreorchis is closely related to leafless Corallorhiza, and (ii) it possesses clustered, multi-branched rhizomes that are often found in fully mycoheterotrophic orchids. Accordingly, we investigated the nutritional modes of O. indica in a Japanese subboreal forest by measuring the 13C and 15N abundances and by community profiling of its mycorrhizal fungi. We found that O. indica mycorrhizal samples (all 12 samples from four individuals) were predominantly colonized by a single OTU of the obligate ectomycorrhizal Tomentella (Thelephoraceae). In addition, the leaves of O. indica were highly enriched in both 13C and 15N compared with those of co-occurring autotrophic plants. It was estimated that O. indica obtained 44.4 ± 6.2% of its carbon from fungal sources. These results strongly suggest that in the Oreorchis-Corallorhiza clade, full mycoheterotrophy evolved after the establishment of partial mycoheterotrophy, rather than through direct shifts from autotrophy.

Tracking long-distance migration of marine fishes using compound-specific stable isotope analysis of amino acids.

The long-distance migrations by marine fishes are difficult to track by field observation. Here, we propose a new method to track such migrations using stable nitrogen isotopic composition at the base of the food web (δ15 NBase ), which can be estimated by using compound-specific isotope analysis. δ15 NBase exclusively reflects the δ15 N of nitrate in the ocean at a regional scale and is not affected by the trophic position of sampled organisms. In other words, δ15 NBase allows for direct comparison of isotope ratios between proxy organisms of the isoscape and the target migratory animal. We initially constructed a δ15 NBase isoscape in the northern North Pacific by bulk and compound-specific isotope analyses of copepods (n = 360 and 24, respectively), and then we determined retrospective δ15 NBase values of spawning chum salmon (Oncorhynchus keta) from their vertebral centra (10 sections from each of two salmon). We then estimated the migration routes of chum salmon during their skeletal growth by using a state-space model. Our isotope tracking method successfully reproduced a known chum salmon migration route between the Okhotsk and Bering seas, and our findings suggest the presence of a new migration route to the Bering Sea Shelf during a later growth stage.

Some mycoheterotrophic orchids depend on carbon from dead wood: novel evidence from a radiocarbon approach.

Mycoheterotrophic plants depend entirely on fungal associations for organic nutrients. While most mycoheterotrophic plants are associated with the mycorrhizal partners of surrounding green plants, some mycoheterotrophs are believed to obtain carbon from decaying litter or dead wood by parasitising saprotrophic fungi, based on culture experiments and 13 C and 15 N isotopic signatures. The carbon age (the time since carbon was fixed from atmospheric CO2 by photosynthesis) can be estimated by measuring the concentration of 14 C arising from the bomb tests of the 1950s and 1960s. Given that mycorrhizal fungi obtain photosynthate from their plant partners, and saprotrophic wood-decaying fungi obtain carbon from older sources, radiocarbon could represent a new and powerful tool to investigate carbon sources of mycoheterotrophic plants. We showed that the Δ14 C values of mycoheterotrophs exploiting ectomycorrhizal fungi were close to 0‰, similar to those of autotrophic plants. By contrast, the Δ14 C values of mycoheterotrophs exploiting saprotrophic fungi ranged from 110.7‰ to 324.8‰, due to the 14 C-enriched bomb carbon from dead wood via saprotrophic fungi. Our study provides evidence supporting that some mycoheterotrophic orchids depend on forest woody debris. Our study also indicates that radiocarbon could be used to predict the trophic strategies of mycoheterotroph-associated fungal symbionts.

Evidence for newly discovered albino mutants in a pyroloid: implication for the nutritional mode in the genus Pyrola.

PREMISE: Difficulties in comparing extremely divergent features in fully mycoheterotrophic plants with those in closely related chlorophyllous plants have complicated attempts to reveal the evolutionary patterns and processes of fully mycoheterotrophic plants. Albino mutants of partially mycoheterotrophic plants, generally observed in Orchidaceae, have provided an ideal model for investigating the evolution of mycoheterotrophy within similar genetic backgrounds. In 2018, we found a putative albino population of Pyrola (Ericaceae). Here we aimed to reveal the identity of the albino pyroloid and confirm its fully mycoheterotrophic status. METHODS: To reveal the putative albino pyroloid's identity, we examined its morphology and sequenced its chloroplast DNA. In addition, we assessed the trophic status of the putative albino pyroloid by analyzing chlorophyll fluorescence, chlorophyll concentration, and natural 13 C and 15 N abundances. RESULTS: We identified albino individuals as P. japonica-otherwise a partially mycoheterotrophic species. We confirmed their albino status by their considerably lower chlorophyll fluorescence and concentrations than those of sympatrically occurring chlorophyllous plants. 13 C abundance in the albino individuals was significantly higher than in the green individuals of P. japonica. CONCLUSIONS: This first report of albino mutants from partially mycoheterotrophic species in angiosperms other than orchids will play a valuable role in further studies focused on mycoheterotrophy. For instance, their δ13 C and δ15 N values represent a reference for fully mycoheterotrophic plants in Pyrola. Our findings also indicate the strong dependence of some leafy Pyrola species on fungal C during their entire life cycle.

Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina, an orchid mainly utilizing saprotrophic rhizoctonia.

The majority of chlorophyllous orchids form mycorrhizal associations with so-called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their 13 C and 15 N abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in 13 C but not so greatly in 15 N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, 13 C levels were depleted and those of 15 N were indistinguishable from the co-occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.

Identification of Phosphorus Sources in a Watershed Using a Phosphate Oxygen Isoscape Approach.

Identifying nonpoint phosphorus (P) sources in a watershed is essential for addressing cultural eutrophication and for proposing best-management solutions. The oxygen isotope ratio of phosphate (δ18OPO4) can shed light on P sources and P cycling in ecosystems. This is the first assessment of the δ18OPO4 distribution in a whole catchment, namely, the Yasu River Watershed in Japan. The observed δ18OPO4 values in the river water varied spatially from 10.3‰ to 17.6‰. To identify P sources in the watershed, we used an isoscape approach involving a multiple-linear-regression model based on land use and lithological types. We constructed two isoscape models, one using data only from the whole watershed and the other using data from the small tributaries. The model results explain 69% and 96% of the spatial variation in the river water δ18OPO4. The lower R2 value for the whole watershed model is attributed to the relatively large travel time for P in the main stream of the lower catchment that can result in cumulative biological P recycling. Isoscape maps and a correlation analysis reveal the relative importance of P loading from paddy fields and bedrock. This work demonstrates the utility of δ18OPO4 isoscape models for assessing nonpoint P sources in watershed ecosystems.

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.

Partial mycoheterotrophy in the leafless orchid Cymbidium macrorhizon.

PREMISE OF THE STUDY: The evolution of full mycoheterotrophy is one of the most interesting topics within plant evolution. The leafless orchid Cymbidium macrorhizon is often assumed to be fully mycoheterotrophic even though it has a green stem and fruit capsule. Here, we assessed the trophic status of this species by analyzing the chlorophyll content and the natural 13 C and 15 N abundance in the sprouting and the fruiting season. METHODS: The chlorophyll content was measured in five sprouting and five fruiting individuals of C. macrorhizon that were co-occurring. In addition, their 13 C and 15 N isotopic signatures were compared with those of neighboring autotrophic and partially mycoheterotrophic reference plants. KEY RESULTS: Fruiting individuals of C. macrorhizon were found to contain a remarkable amount of chlorophyll compared to their sprouting counterparts. In addition, the natural abundance of 13 C in the tissues of the fruiting plants was slightly depleted relative to the sprouting ones. Linear two-source mixing model analysis revealed that fruiting C. macrorhizon plants obtained approximately 73.7 ± 2.0% of their total carbon from their mycorrhizal fungi when the sprouting individuals were used as the 100% carbon gain standard. CONCLUSIONS: Our results indicated that despite its leafless status, fruiting plants of C. macrorhizon were capable of fixing significant quantities of carbon. Considering the autotrophic carbon gain increases during the fruiting season, its photosynthetic ability may contribute to fruit and seed production. These results indicate that C. macrorhizon should, therefore, be considered a partially mycoheterotrophic species rather than fully mycoheterotrophic, at least during the fruiting stage.

Differential Responses of Two Ecologically Similar Case-Bearing Caddisfly Species to a Fish Chemical Cue: Implications for a Coexistence Mechanism.

The mechanisms for the coexistence of multiple species occupying the same ecological niche are often puzzling. Predator effects on competitively superior species is one possible mechanism. In this study, we tested whether the presence of size-selective predators (fishes) acts as a mechanism for the coexistence of two species of case-bearing caddisfly larvae, Perissoneura paradoxa and Psilotreta kisoensis (Odontoceridae, Trichoptera). The larvae of these two species have similar ecological and life history traits except their body size, and they have been found to coexist only in habitats shared with predatory fishes. Experiments on intra and interspecific competition revealed that the larger Pe. paradoxa always outcompeted the smaller Ps. kisoensis in the absence of predatory fishes, suggesting that Pe. paradoxa performed intra-guild predation on Ps. kisoensis. We also conducted experiments to examine how strongly each of these species responded in terms of case repair with/without a predator chemical cue after their cases were partly dismantled. Perissoneura paradoxa exhibited a stronger case repair response in the presence of a predator chemical cue than that exhibited by Ps. kisoensis, suggesting that Pe. paradoxa is more vulnerable to fish predation, probably because their body size is in the preferred prey range of fishes. We suggest that the presence of predators works in the favor of smaller, subordinate species through size-selective predator effects, enabling these two competitive species to coexist in the same habitat.

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.

A comparison of freeze-drying and oven-drying preparation methods for bulk and compound-specific carbon stable isotope analyses: examples using the benthic macroinvertebrates Stenopsyche marmorata and Epeorus latifolium.

RATIONALE: Carbon stable isotope analysis of bulk samples and fatty acids is an established method for tracing carbon flow pathways and reconstructing trophic interactions, but there is no consensus on which sample drying method should be used for sample preparation. The aim of this study was to determine if freeze-drying and oven-drying treatments used to prepare samples of the benthic macroinvertebrates Stenopsyche marmorata and Epeorus latifolium for bulk and fatty-acid-specific carbon stable isotope analysis yield different isotopic ratio values. METHODS: Five individuals each from two species were split in half; one half was freeze-dried and the other half was oven-dried. The samples were ground and the δ(13)C values of the bulk samples and eight fatty acids were measured following combustion using an isotope ratio mass spectrometer coupled to an elemental analyzer or gas chromatography system. RESULTS: The mean difference in the bulk and fatty acid δ(13)C values between freeze-dried and oven-dried samples was small (≤0.1‰ in both cases), although relatively large variations were observed in individual fatty-acid-specific δ(13)C values (maximum of ≤0.9 ‰). There were no significant differences in either bulk sample or fatty-acid-specific δ(13)C values between freeze-dried or oven-dried samples of the same species. CONCLUSIONS: Freeze-drying and oven-drying are equally acceptable methods for preparing freshly caught S. marmorata and E. latifolium samples for bulk and fatty-acid-specific carbon stable isotope analyses.