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.

Strontium isotope analysis of otoliths reveals differences in the habitat salinity among three sympatric stickleback species of the genus Pungitius.

The analysis of otolith Sr isotope ratios (87Sr/86Sr) is a powerful method to study fish migration in freshwater areas. However, few studies have applied this method to study fish movement in brackish-water environments. Furthermore, despite the fact that habitat differentiation has been shown to drive genetic differentiation and reproductive isolation among stickleback fish, no studies have used the otolith 87Sr/86Sr ratios to analyze habitat differentiation between stickleback ecotypes and species. In this study, we analyzed the otolith 87Sr/86Sr ratios of three sympatric stickleback species of the genus Pungitius in the Shiomi River on Hokkaido Island, Japan: P. tymensis, the brackish-water type of the P. pungitius-P. sinensis complex, and the freshwater type of the P. pungitius-P. sinensis complex. First, we created a mixing equation to depict the relationship between habitat salinity and the 87Sr/86Sr ratios of river water. We found that the otolith 87Sr/86Sr ratios differed significantly among the three species, indicating that the three species utilize habitats with different salinities: P. tymensis and the brackish-water type inhabit freshwater and brackish-water environments, respectively, with the freshwater type using intermediate habitats. In addition, we found that some freshwater individuals moved to habitats with higher salinities as they grew. Our study demonstrates that the analysis of otolith 87Sr/86Sr ratios is a useful method for studying the habitat use of fish in brackish-water environments and habitat differentiation among closely related sympatric and parapatric species.

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.

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.

Diagnosing underdetermination in stable isotope mixing models.

Stable isotope mixing models (SIMMs) provide a powerful methodology for quantifying relative contributions of several sources to a mixture. They are widely used in the fields of ecology, geology, and archaeology. Although SIMMs have been rapidly evolved in the Bayesian framework, the underdetermination of mixing space remains problematic, i.e., the estimated relative contributions are incompletely identifiable. Here we propose a statistical method to quantitatively diagnose underdetermination in Bayesian SIMMs, and demonstrate the applications of our method (named ß-dependent SIMM) using two motivated examples. Using a simulation example, we showed that the proposed method can rigorously quantify the expected underdetermination (i.e., intervals of ß-dependent posterior) of relative contributions. Moreover, the application to the published field data highlighted two problematic aspects of the underdetermination: 1) ordinary SIMMs was difficult to quantify underdetermination of each source, and 2) the marginal posterior median was not necessarily consistent with the joint posterior peak in the case of underdetermination. Our study theoretically and numerically confirmed that ß-dependent SIMMs provide a useful diagnostic tool for the underdetermined mixing problem. In addition to ordinary SIMMs, we recommend reporting the results of ß-dependent SIMMs to obtain a biologically feasible and sound interpretation from stable isotope data.

Evaluation of origin-depended nitrogen input through atmospheric deposition and its effect on primary production in coastal areas of western Kyusyu, Japan.

Long term monitoring of atmospheric wet and dry depositions and associated nutrients fluxes was conducted on the coast of Japan facing the East China Sea continuously for 1 year and 2 months, with the origin of air mass investigated based on isotope analyses (Sr, Nd, and NO3). During the same period, intensive observations of ocean conditions and the chemical composition of sinking particles collected using sediment traps were conducted to investigate the effects of atmospheric deposition-derived nutrients on phytoplankton blooms. Dry-deposition-derived nutrient inputs to the surface ocean were larger during autumn to spring than in summer due to the effect of continental air mass occasionally carrying Asian dust (yellow sand). However, these nutrients fluxes were limited (1.1-1.5 mg-N m-2 day-1 on average) and didn't appear to cause phytoplankton blooms through the year. Although average dissolved inorganic nitrogen (DIN) concentrations in rainwater were lower in oceanic air masses compared to continental air masses, wet-deposition-derived nutrient inputs to the surface ocean on rainy days during the summer (26.0 mg-N m-2 day-1 on average) were large due to higher precipitation from oceanic air masses. Wet-deposition-derived nutrients significantly increased nutrient concentrations in the surface ocean and seemed to cause phytoplankton blooms in the warm rainy season when nutrients in the surface were depleted due to increased stratification. The increase in phytoplankton biomass was reflected in increased particle sinking into the bottom layer, as well as changing chemical characteristics. The supply of flesh phytoplankton-derived labile organic matter into the bottom layer could be expected to promote rapid bacterial decomposition and contribute to the formation of hypoxic water masses in early summer when the ocean was strongly stratified. Atmospheric deposition-derived nutrients in East Asia will have important impacts on not only the oligotrophic outer ocean but also surrounding coastal areas in the warm rainy season.

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.

Use of radiocarbon for assessing the mycorrhizal status of mycoheterotrophic plants.

Mycoheterotrophic plants are non-photosynthetic plants that obtain nutrients from fungi. Even though most of these plants are associated with the mycorrhizal partners of surrounding photosynthetic plants, recent studies have suggested that some mycoheterotrophic orchids indirectly obtain carbon from decaying organic matter through associations with saprotrophic fungi. However, such suggestions have been based primarily on indirect evidence, such as the 13C and 15N abundances of fungi and plants. It was recently reported that some mycoheterotrophs yield elevated Δ14C values, owing to the indirect acquisition of 14C-enriched bomb carbon from dead wood. The approach was based on the anthropogenic change of Δ14C values; atmospheric CO2 were globally elevated by nuclear weapons testing in the 1950s and early 1960s, but have steadily declined since its peak after the atmospheric nuclear test ban treaty of 1963. The study has provided novel evidence that mycoheterotrophic plants can exploit both mycorrhizal and saprotrophic fungi. We suggest that the radiocarbon analysis is also useful for investigating the nutritional modes of mixotrophic plants as well as for investigating whether the recruitment of wood-decaying fungi into novel mycorrhizal partnerships preceded the evolution of full mycoheterotrophy.

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.

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.

Trust me? Consumer trust in expert information on food product labels.

Food product labels can provide consumers with rich, specific, expert-certified product information. However, sources of label information differ. How do consumers then evaluate label trustworthiness of expert labels in comparison to other commonly used label types? We present results from a representative online survey (N = 10,000) of consumers in Japan, the USA, Germany, China and Thailand using professionally designed labels for four food types (milk, honey, oil, wine) and five different sources of food information (farmers, government/administration, producer associations, experts, and consumers). We tested label legibility through identification of the label information source and asked respondents to evaluate the trustworthiness of labels using a six-scale instrument ranging from overall label trust to purchase intent. Results show that label legibility varied between countries, with expert labels scoring lowest. Nevertheless, respondents correctly identifying all label information sources chose expert labels as the most or second-most trustworthy across all countries and food types, while consumer labels scored low. Demographic factors exhibited weak influence. Results suggest expert labels might play an important role as trusted sources of information in an increasingly complex global food system. Finally, we consider the implications of the study for a potential institutionalization of expert labels based on the Japanese context.

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.

Downhill seed dispersal by temperate mammals: a potential threat to plant escape from global warming.

Vertical seed dispersal, i.e. seed dispersal towards a higher or lower altitude, is considered a critical process for plant escape from climate change. However, studies exploring vertical seed dispersal are scarce, and thus, its direction, frequency, and mechanisms are little known. In the temperate zone, evaluating vertical seed dispersal of animal-dispersed plants fruiting in autumn and/or winter is essential considering the dominance of such plants in temperate forests. We hypothesized that their seeds are dispersed towards lower altitudes because of the downhill movement of frugivorous animals following the autumn-to-winter phenology of their food plants which proceeds from the mountain tops to the foot in the temperate zone. We evaluated the vertical seed dispersal of the autumn-fruiting wild kiwi, Actinidia arguta, which is dispersed by temperate mammals. We collected dispersed seeds from mammal faeces in the Kanto Mountains of central Japan and estimated the distance of vertical seed dispersal using the oxygen isotope ratios of the dispersed seeds. We found the intensive downhill seed dispersal of wild kiwi by all seed dispersers, except the raccoon dog (bear: mean -393.1 m; marten: -245.3 m; macaque: -98.5 m; and raccoon dog: +4.5 m). Mammals with larger home ranges dispersed seeds longer towards the foot of the mountains. Furthermore, we found that seeds produced at higher altitudes were dispersed a greater distance towards the foot of the mountains. Altitudinal gradients in autumn-to-winter plant phenology and other mountain characteristics, i.e. larger surface areas and more attractive human crops at lower altitudes compared to higher altitudes, were considered drivers of downhill seed dispersal via animal movement. Strong downhill seed dispersal by mammals suggests that populations of autumn-to-winter fruiting plants dispersed by animals may not be able to sufficiently escape from current global warming in the temperate zone.