Ten facts about land systems for sustainability.

Land use is central to addressing sustainability issues, including biodiversity conservation, climate change, food security, poverty alleviation, and sustainable energy. In this paper, we synthesize knowledge accumulated in land system science, the integrated study of terrestrial social-ecological systems, into 10 hard truths that have strong, general, empirical support. These facts help to explain the challenges of achieving sustainability in land use and thus also point toward solutions. The 10 facts are as follows: 1) Meanings and values of land are socially constructed and contested; 2) land systems exhibit complex behaviors with abrupt, hard-to-predict changes; 3) irreversible changes and path dependence are common features of land systems; 4) some land uses have a small footprint but very large impacts; 5) drivers and impacts of land-use change are globally interconnected and spill over to distant locations; 6) humanity lives on a used planet where all land provides benefits to societies; 7) land-use change usually entails trade-offs between different benefits-"win-wins" are thus rare; 8) land tenure and land-use claims are often unclear, overlapping, and contested; 9) the benefits and burdens from land are unequally distributed; and 10) land users have multiple, sometimes conflicting, ideas of what social and environmental justice entails. The facts have implications for governance, but do not provide fixed answers. Instead they constitute a set of core principles which can guide scientists, policy makers, and practitioners toward meeting sustainability challenges in land use.

RNA-seq analysis to identify genes related to resting egg production of panarctic Daphnia pulex.

BACKGROUND: The genus Daphnia switches its reproductive mode from subitaneous egg production to resting egg production in response to environmental stimuli. Although this life history trait is essential for surviving unsuitable environments, the molecular mechanism of resting egg production is little understood. In this study, we examined genes related to induction of resting egg production using two genotypes of panarctic Daphnia pulex, the JPN1 and JPN2 lineages, which differ genetically in the frequency of resting egg production. We reared these genotypes under high and low food levels. At the high food level, individuals of both genotypes continually produced subitaneous eggs, whereas at the low food level, only the JPN2 genotype produced resting eggs. Then, we performed RNA-seq analysis on specimens of three instars, including before and after egg production. RESULTS: These results showed that expressed genes differed significantly between individuals grown under high and low food levels and among individuals of different instars and genotypes. Among these differentially expressed genes (DEGs), we found 16 that changed expression level before resting egg production. Some of these genes showed high-level expression only before resting egg production and one gene was an ortholog of bubblegum (bgm), which is reportedly up-regulated before diapause in bumblebees. According to gene ontology (GO) enrichment analysis, one GO term annotated as long-chain fatty acid biosynthetic process was enriched among these 16 genes. In addition, GO terms related to glycometabolism were enriched among down-regulated genes of individuals holding resting eggs, compared to those before resting egg production. CONCLUSIONS: We found candidate genes highly expressed only before resting egg production. Although functions of candidate genes found in this study have not been reported previously in Daphnia, catabolism of long-chain fatty acids and metabolism of glycerates are related to diapause in other organisms. Thus, it is highly probable that candidate genes identified in this study are related to the molecular mechanism regulating resting egg production in Daphnia.

Divergence of the Host-Associated Microbiota with the Genetic Distance of Host Individuals Within a Parthenogenetic Daphnia Species.

The taxonomic composition of the microbiota in the gut and epidermis of animals is known to vary among genetically and physiologically different host individuals within the same species. However, it is not clear whether the taxonomic composition diverges with increasing genetic distance of the host individuals. To unveil this uncertainty, we compared the host-associated microbiota among the genotypes within and between genetically distant lineages of parthenogenetic Daphnia cf. pulex across different physiological states, namely, well-fed, starved, and dead. Metagenomic analysis with 16S rRNA showed that, regardless of the host genotypes, diversity of the host-associated microbiota was high when the host individuals were fed food and gradually decreased when they were starved until they died. However, the difference in the host-associated microbiota, that is, ß-diversity, was significant among the genotypes within and between the host lineages when they were fed. Although some bacteria in the microbiota, such as Limnohabitans, Rhodococcus, and Aeromicrobium, were found abundantly and commonly in all host genotypes; others, such as those of Holosoporacea, were found only in the genotypes of a specific lineage. Accordingly, the ß-diversity tended to increase with increasing genetic distance of the host individuals. These results support an idea that the host-associated microbiota diverged with genetic divergence in the host species and that at least some bacteria are highly dependent on the genetically specific metabolites produced by the host individuals.

Evolution of asexual Daphnia pulex in Japan: variations and covariations of the digestive, morphological and life history traits.

BACKGROUND: Several genetic lineages of obligate parthenogenetic Daphnia pulex, a common zooplankton species, have invaded Japan from North America. Among these, a lineage named JPN1 is thought to have started colonization as a single genotype several hundred to thousand years ago and subsequently produced many genotypes in Japan. To examine the phenotypic variations due to ecological drivers diverging the genotypes in new habitats, we measured heritability and variation in 17 traits, including life history, morphology and digestive traits, and the genetic distance among the D. pulex JPN1 genotypes in Japan. RESULTS: We found that most of the traits measured varied significantly among the genotypes and that heritability was highest in the morphological traits, followed by the digestive and life history traits. In addition, 93% of the variation in these traits was explained by the first three components in the principal component analysis, implying that variation of these heritable traits is not random but rather converged into a few directions. These relations among traits revealed the potential importance of predation pressures and food conditions as factors for diverging and selecting different genotypes. However, the magnitude of the difference in any single trait group did not correlate with the genetic distance. CONCLUSIONS: Our findings show that the divergent traits evolved within D. pulex JPN1 lineage without genetic recombination, since their ancestral clone invaded Japan. Large variations and covariations of the phenotypic traits, irrespective of the genetic distance among the genotypes, support the view that the invasive success of D. pulex JPN1 was promoted by a genetic architecture that allowed for large phenotypic variations with a limited number of functionally important mutations without recombination.

Understanding the stoichiometric limitation of herbivore growth: the importance of feeding and assimilation flexibilities.

Ecological stoichiometry suggests that herbivore growth is limited by phosphorus when this element in the diet is < 8.6 µg P mg C-1 (C : P atomic ratio > 300). However, in nature, it is not necessarily related to the relative phosphorus content in diets. This may be the result of complex feeding and assimilation responses to diets. We examined these possibilities using herbivorous plankton fed mono-specific and mixed algae varying in phosphorus content of 1.6 to 8.1 µg P mg C-1 . The herbivores showed a 10-fold growth rate difference among the diets. Growth rates related poorly with phosphorus content in the diets (r2  = 0.07), better with P ingestion rate (r2  = 0.41) and best with phosphorus assimilation rate (r2  = 0.69). Inclusion of assimilation rates for carbon and fatty acids increased 7% of the explained growth variance. These results indicate that the feeding and assimilation flexibilities play pivotal roles in acquiring a deficient element and in regulating growth rate.

A shady phytoplankton paradox: when phytoplankton increases under low light.

Light is a fundamental driver of ecosystem dynamics, affecting the rate of photosynthesis and primary production. In spite of its importance, less is known about its community-scale effects on aquatic ecosystems compared with those of nutrient loading. Understanding light limitation is also important for ecosystem management, as human activities have been rapidly altering light availability to aquatic ecosystems. Here we show that decreasing light can paradoxically increase phytoplankton abundance in shallow lakes. Our results, based on field manipulation experiments, field observations and models, suggest that, under competition for light and nutrients between phytoplankton and submersed macrophytes, alternative stable states are possible under high-light supply. In a macrophyte-dominated state, as light decreases phytoplankton density increases, because macrophytes (which effectively compete for nutrients released from the sediment) are more severely affected by light reduction. Our results demonstrate how species interactions with spatial heterogeneity can cause an unexpected outcome in complex ecosystems. An implication of our findings is that partial surface shading for controlling harmful algal bloom may, counterintuitively, increase phytoplankton abundance by decreasing macrophytes. Therefore, to predict how shallow lake ecosystems respond to environmental perturbations, it is essential to consider effects of light on the interactions between pelagic and benthic producers.

Rediscovery after Almost 120 Years: Morphological and Genetic Evidence Supporting the Validity of Daphnia mitsukuri (Crustacea: Cladocera).

We examined the morphology of Daphnia individuals maintained in our laboratory for several years, originally collected in Lake Inbanuma, Chiba, Japan. We determined partial sequences of the mitochondrial cytochrome c oxidase subunit I and 12S rRNA genes from specimens in the cultured material. These animals are morphologically similar to D. obtusa Kurz, 1874 , but genetically distinct from this species. Our detailed observation shows that the morphological characteristics in the female and male individuals of our material are highly congruent with those of D. mitsukuri Ishikawa, 1896 , which has not been identified positively for more than 120 years since its original description, with its taxonomic identity having been questioned for almost 90 years. Based on our morphological and genetic data, we conclude that D. mitsukuri should be regarded as a taxonomically valid species. A search among public DNA sequence databases suggests D. mitsukuri is also distributed in China, although these Chinese sequences have been labeled as 'Daphnia pulex', representing misidentification.

Will Invertebrates Require Increasingly Carbon-Rich Food in a Warming World?

Elevated temperature causes metabolism and respiration to increase in poikilothermic organisms. We hypothesized that invertebrate consumers will therefore require increasingly carbon-rich diets in a warming environment because the increased energetic demands are primarily met using compounds rich in carbon, that is, carbohydrates and lipids. Here, we test this hypothesis using a new stoichiometric model that has carbon (C) and nitrogen (N) as currencies. Model predictions did not support the hypothesis, indicating instead that the nutritional requirements of invertebrates, at least in terms of food quality expressed as C∶N ratio, may change little, if at all, at elevated temperature. Two factors contribute to this conclusion. First, invertebrates facing limitation by nutrient elements such as N have, by default, excess C in their food that can be used to meet the increased demand for energy in a warming environment, without recourse to extra dietary C. Second, increased feeding at elevated temperature compensates for the extra demands of metabolism to the extent that, when metabolism and intake scale equally with temperature (have the same Q10), the relative requirement for dietary C and N remains unaltered. Our analysis demonstrates that future climate-driven increases in the C∶N ratios of autotroph biomass will likely exacerbate the stoichiometric mismatch between nutrient-limited invertebrate grazers and their food, with important consequences for C sequestration and nutrient cycling in ecosystems.

Geographical variation of bacterial and ciliophoran communities in tidal flats in a continental archipelago.

In tidal flats, which are located at the transition zone between terrestrial and marine ecosystems, environmental factors such as temperature, sediment particle size, and tidal range exhibit geographic variation. Accordingly, the composition and structure of the microbial communities in the tidal flats are likely to vary in geographically different habitats. To clarify these differences with environmental factors causing them, we analyzed microbial communities consisting of bacteria and ciliates in sediments collected from nine tidal flats in geographical diverse region from Hokkaido to Kagoshima, Japan. The results confirmed that the community structures of bacteria and ciliophora in tidal flat sediments differed at the geographical scale of the Japanese archipelago. However, the variation could not be explained by the physical distance between the tidal flats nor by the differences in the trophic conditions among the tidal flats. Instead, the OTU richness of both the bacterial and ciliophoran communities was significantly related to the tidal range. The results also showed that bacteria and ciliophora tended to form similar communities among the tidal flats with similar median particle sizes. Furthermore, ciliophoran communities were similar among the tidal flats with similar bacterial communities. The results suggest that bacteria and ciliophora interact each other through trophic relationships or physical and chemical processes in the sediment habitats.

Assessment of toxic effects of imidacloprid on freshwater zooplankton: An experimental test for 27 species.

The neonicotinoid pesticide imidacloprid has been used worldwide since 1992. As one of the most important chemicals used in pest control, there have been concerns that its run-off into rivers and lakes could adversely affect aquatic ecosystems, where zooplankton play a central role in the energy flow from primary to higher trophic levels. However, studies assessing the effects of pesticides at the species level have relied on a Daphnia-centric approach, and no studies have been conducted using species-level assessments on a broad range of zooplankton taxa. In the present study, we therefore investigated the acute toxicity of imidacloprid on 27 freshwater crustacean zooplankton (18 cladocerans, 3 calanoid copepods and 6 cyclopoid copepods). The experiment showed that a majority of calanoid copepods and cladocerans were not affected at all by imidacloprid, with the exception of one species each of Ceriodaphnia and Diaphasoma, while all six cyclopoid copepods showed high mortality rates, even at concentrations of imidacloprid typically found in nature. In addition, we found a remarkable intra-taxonomic variation in susceptibility to this chemical. As many cyclopoid copepods are omnivorous, they act as predators as well as competitors with other zooplankton. Accordingly, their susceptibility to imidacloprid is likely to cause different responses at the community level through changes in predation pressure as well as changes in competitive interactions. The present results demonstrate the need for species-level assessments of various zooplankton taxa to understand the complex responses of aquatic communities to pesticide disturbance.

Contrasting population genetic structure of three semi-terrestrial brachyuran crabs on the coast of the Japanese archipelago.

Anthropogenic activities have reduced ecotones between the ocean and land, which is likely to threaten the population of brackish-water brachyuran crabs. To assess the current status of these crabs, we examine the population genetic structures of three semi-terrestrial brachyuran crabs widely distributed along the coast of the Japan and to clarify factors determining their genetic structures. We collected 184 Orisarma dehaani, 252 Chiromantes haematocheir, and 151 Helice tridens crabs from 36 localities of the Japanese archipelago. Genome-wide SNP data from these crabs were analyzed using MIG-seq. Bayesian clustering of STRUCTURE and DAPC analysis were used to identify genetically disturbed populations and to visualize genetic differentiation between local populations. Genetic population structure showed clear differentiation between populations on the Pacific coast of the Tohoku region and on other Japanese coasts in O. dehaani, but not in C. haematocheir or H. tridens. The inbreeding coefficient of O. dehaani was significantly higher on the Pacific coast of the Tohoku region compared to other Japanese coasts. C. haematocheir and H. tridens had homogeneous genetic structures along the Japanese coast, but showed genetic differentiation of a local population at their range limits. Thus, O. dehaani showed little gene flow and clear genetic differentiation between populations in the Tohoku Pacific region and those on other Japanese coasts due to ocean currents. Although such a regional differentiation was not found in C. haematocheir and H. tridens, one population of C. haematocheir was genetically isolated at the edge of its distribution range and likely vulnerable to environmental changes.

Stoichiometry of carbon, nitrogen, and phosphorus released from the leaf litter of various temperate tree species.

Dissolved organic matter and inorganic nutrients released from forest leaf litter through leaching are the important energy and nutrient sources that support the production of aquatic food webs. Leaf litter-derived dissolved organic carbon (DOC) is a critical energy source for aquatic heterotrophic microbes, and inorganic nitrogen and phosphorus can enhance primary production. In this study, we experimentally measured the release efficiencies and amounts of DOC, total dissolved nitrogen (TDN), and total dissolved phosphorus (TDP) of the leaf litter from 11 temperate tree species by soaking the leaf litter in water for 28 days. We found that the maximal release efficiency (% of element released per estimated mass of the element) was the highest for P and lowest for N. These efficiencies were species-specific. Additionally, the DOC:TDP, DOC:TDN, and TDN:TDP ratios varied among the leachate of different leaf litter species. DOC:TDP increased with the C:P ratio in leaf litter biomass but is considerably lower; TDN:TDP was lower than the N:P ratio in leaf litter biomass as well; DOC:TDN ratio was higher than the C:N ratio in leaf litter biomass. These results suggest that the ratios of DOC to dissolved N and P nutrients released into water are related to, but not the same as, the stoichiometry of leaf litter biomass. Based on these findings, we concluded that changes in the vegetations with different leaf litter stoichiometry can alter the relative importance of detrital and grazing food chains in aquatic ecosystems.

Dissolved organic carbon as major environmental factor affecting bacterioplankton communities in mountain lakes of eastern Japan.

Relationships between environmental factors and bacterial communities were investigated in 41 freshwater lakes located in mountainous regions of eastern Japan. Bacterioplankton community composition (BCC) was determined by polymerase chain reaction-denaturing gradient gel electrophoresis of the 16S rRNA gene and then evaluated on the basis of physicochemical and biological variables of the lakes. Canonical correspondence analysis revealed that BCC of oligotrophic lakes was significantly influenced by dissolved organic carbon (DOC) content, but its effect was not apparent in the analysis covering all lakes including mesotrophic and eutrophic ones. The generalized linear model showed the negative association of DOC on the taxon richness of bacterioplankton communities. DOC was positively correlated with the catchment area per lake volume, suggesting that a large fraction of DOC supplied to the lake was derived from terrestrial sources. These results suggest that allochthonous DOC has a significant effect on bacterioplankton communities especially in oligotrophic lakes. The genus Polynucleobacter was detected most frequently. The occurrence of Polynucleobacter species was positively associated with DOC and negatively associated with total phosphorus (TP) levels. In addition, TP had a stronger effect than DOC, suggesting that oligotrophy is the most important factor on the occurrence of this genus.

Invasions of an obligate asexual daphnid species support the nearly neutral theory.

To verify the "nearly neutral theory (NNT)," the ratio of nonsynonymous to synonymous substitutions (dN/dS) was compared among populations of different species. To determine the validity of NNT, however, populations that are genetically isolated from each other but share the same selection agents and differ in size should be compared. Genetically different lineages of obligate asexual Daphnia pulex invading Japan from North America are an ideal example as they satisfy these prerequisites. Therefore, we analyzed the whole-genome sequences of 18 genotypes, including those of the two independently invaded D. pulex lineages (JPN1 and JPN2) and compared the dN/dS ratio between the lineages. The base substitution rate of each genotype demonstrated that the JPN1 lineage having a larger distribution range diverged earlier and thus was older than the JPN2 lineage. Comparisons of the genotypes within lineages revealed that changes in dN/dS occurred after the divergence and were larger in the younger lineage, JPN2. These results imply that the JPN1 lineage has been more effectively subjected to purification selections, while slightly deteriorating mutations are less purged in JPN2 with smaller population size. Altogether, the lineage-specific difference in the dN/dS ratio for the obligate asexual D. pulex was well explained by the NNT.

The long-term consequences of hybridization between the two Daphnia species, D. galeata and D. dentifera, in mature habitats.

BACKGROUND: Ecological specializations such as antipredator defense can reinforce morphological and distributional divergence within hybridizing species. Two hybridizing species of Daphnia (D. galeata and D. dentifera) are distributed in both Japan and North America; however, these populations have a longer history in Japan than in North America due to the differing impact of the last glaciation on these two regions. We tested the hypothesis that this longer coexistence in Japan would lead to extensive genetic admixture in nuclear and mitochondrial DNA whilst the distinct morphological traits and distributional patterns would be maintained. RESULTS: The high level of correspondence among morphological traits, distribution, and mitochondrial and nuclear DNA types for the specimens with D. dentifera mtDNA indicated that the species distinction has been maintained. However, a discordance between mtDNA and nuclear ITS-1 types was observed for most specimens that had D. galeata mtDNA, consistent with the pattern seen between the two species in North America. This observation suggests nuclear introgression from D. dentifera into D. galeata without mitochondrial introgression. CONCLUSIONS: The separation of morphological traits and distribution ranges of the two hybridizing species in Japan, as well as in North America, has been maintained, despite large differences in climatic and geographical histories of these two regions. Variations in environmental factors, such as predation pressure, might affect maintenance of the distribution, although the further studies are needed to confirm this.

Microbial communities developing within bulk sediments under fish carcasses on a tidal flat.

Animal carcasses are often brought into tidal flats where they are at the boundary between terrestrial and marine ecosystems. Since these carcasses act as microhabitats with large amounts of energy and nutrients, they likely develop unique bacterial assemblages in the ambient sediment, which in turn may stimulate colonization of other organisms such as protozoans. However, little is known about the microbial assemblages colonized in sediment around animal carcasses in the tidal zone. Herein we examined the bacterial and ciliophoran assemblages developed in association with fish carcasses by incubating the carcasses in the Higashiyachi tidal flat (Sendai, Japan). We collected sediment samples at 2, 9, and 42 days of incubation and analyzed the bacterial and ciliophoran assemblages by 16S and 18S rRNA gene amplicon sequencing. We observed significant differences in the composition and relative abundance of bacterial and ciliophoran operational taxonomic units (OTUs) between the sediments with and without the carcasses. Our analyses suggest that these unique assemblages were created through the direct effects of the carcass and indirect effects through interactions between bacteria and ciliophorans. These results also suggest that animal carcasses developed a temporally unique microbial food web in the sediments close to the carcasses, although it disappeared for several weeks.

Long-term dynamics of a cladoceran community from an early stage of lake formation in Lake Fukami-ike, Japan.

An increase in nutrient levels due to eutrophication has considerable effects on lake ecosystems. Cladocerans are intermediate consumers in lake ecosystems; thus, they are influenced by both the bottom-up and top-down effects that occur as eutrophication progresses. The long-term community succession of cladocerans and the effects cladocerans experience through the various eutrophication stages have rarely been investigated from the perspective of the early-stage cladoceran community assemblage during lake formation. In our research, long-term cladoceran community succession was examined via paleolimnological analysis in the currently eutrophic Lake Fukami-ike, Japan. We measured the concentration of total phosphorus and phytoplankton pigments and counted cladoceran and other invertebrate subfossils in all layers of collected sediment cores, and then assessed changes in the factors controlling the cladoceran community over a 354-year period from lake formation to the present. The cladoceran community consisted only of benthic taxa at the time of lake formation. When rapid eutrophication occurred and phytoplankton increased, the benthic community was replaced by a pelagic community. After further eutrophication, large Daphnia and high-order consumers became established. The statistical analysis suggested that bottom-up effects mainly controlled the cladoceran community in the lake's early stages, and the importance of top-down effects increased after eutrophication occurred. Total phosphorus and phytoplankton pigments had positive effects on pelagic Bosmina, leading to the replacement of the benthic cladoceran community by the pelagic one. In contrast, the taxa established posteutrophication were affected more by predators than by nutrient levels. A decrease in planktivorous fish possibly allowed large Daphnia to establish, and the subsequent increase in planktivorous fish reduced the body size of the cladoceran community.

A unified framework for herbivore-to-producer biomass ratio reveals the relative influence of four ecological factors.

The biomass ratio of herbivores to primary producers reflects the structure of a community. Four primary factors have been proposed to affect this ratio, including production rate, defense traits and nutrient contents of producers, and predation by carnivores. However, identifying the joint effects of these factors across natural communities has been elusive, in part because of the lack of a framework for examining their effects simultaneously. Here, we develop a framework based on Lotka-Volterra equations for examining the effects of these factors on the biomass ratio. We then utilize it to test if these factors simultaneously affect the biomass ratio of freshwater plankton communities. We found that all four factors contributed significantly to the biomass ratio, with carnivore abundance having the greatest effect, followed by producer stoichiometric nutrient content. Thus, the present framework should be useful for examining the multiple factors shaping various types of communities, both aquatic and terrestrial.

Effects of drainage-basin geomorphology on insectivorous bird abundance in temperate forests.

Interfaces between terrestrial and stream ecosystems often enhance species diversity and population abundance of ecological communities beyond levels that would be expected separately from both the ecosystems. Nevertheless, no study has examined how stream configuration within a watershed influences the population of terrestrial predators at the drainage-basin scale. We examined the habitat and abundance relationships of forest insectivorous birds in eight drainage basins in a cool temperate forest of Japan during spring and summer. Each basin has different drainage-basin geomorphology, such as the density and frequency of stream channels. In spring, when terrestrial arthropod prey biomass is limited, insectivorous birds aggregated in habitats closer to streams, where emerging aquatic prey was abundant. Nevertheless, birds ceased to aggregate around streams in summer because terrestrial prey became plentiful. Watershed-scale analyses showed that drainage basins with longer stream channels per unit area sustained higher densities of insectivorous birds. Moreover, such effects of streams on birds continued from spring through summer, even though birds dispersed out of riparian areas in the summer. Although our data are from only a single year, our findings imply that physical modifications of stream channels may reduce populations of forest birds; thus, they emphasize the importance of landscape-based management approaches that consider both stream and forest ecosystems for watershed biodiversity conservation.

River Metabolism along a Latitudinal Gradient across Japan and in a global scale.

Since temperature is a key factor affecting photosynthetic and respiration rates, the rates of gross primary production (GPP) and ecosystem respiration (ER) are expected to be lower for rivers at higher latitudes, while the net ecosystem production (NEP) rate likely decrease in rivers at lower latitude due to higher sensitivity of ER to temperature compared with GPP. To examine these possibilities, we estimated the ecosystem metabolism of 30 rivers located from 43.03°N to 32.38°N in Japan during summer using a Bayesian model with hourly changes in dissolved oxygen concentrations. In addition, we examined latitudinal trends of GPP, ER and NEP in a global scale by compiling and analyzing river metabolic data estimated in previous studies. Our analysis showed that both GPP and ER tended to increase with latitude, although these rates were positively related to water temperature in Japanese rivers. Global dataset of GPP and ER also showed increasing trend towards higher latitude. In addition, contrary to our initial expectations, NEP decreased with latitude and most rivers were net heterotrophic at both regional (Japanese rivers) and global scales. These results imply that the latitudinal temperature effect on river metabolism is masked by other factors not examined in this study, such as land use in the watershed, which play pivotal roles in explaining the latitudinal variation of river metabolism.