Ecology

1.Jane, S. F. et al. Distance, flow and PCR inhibition: eDNA dynamics in two headwater streams. Mol. Ecol. Resour. 15, 216–227 (2015).CAS 
Article 

Google Scholar 
2.Thomsen, P. F. & Willerslev, E. Environmental DNA: An emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv. 183, 4–18 (2015).Article 

Google Scholar 
3.Valentini, A. et al. Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding. Mol. Ecol. 25, 929–942 (2016).CAS 
Article 

Google Scholar 
4.Harper, L. R. et al. Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of great crested newt (Triturus cristatus). Ecol. Evol. 8, 6330–6341 (2018).Article 

Google Scholar 
5.Ficetola, G. F. et al. Replication levels, false presences and the estimation of the presence/absence from eDNA metabarcoding data. Mol. Ecol. Resour. 15, 543–556 (2015).CAS 
Article 

Google Scholar 
6.Willoughby, J. R., Wijayawardena, B. K., Sundaram, M., Swihart, R. K. & DeWoody, J. A. The importance of including imperfect detection models in eDNA experimental design. Mol. Ecol. Resour. 16, 837–844 (2016).CAS 
Article 

Google Scholar 
7.Burian, A. et al. Improving the reliability of eDNA data interpretation. Mol. Ecol. Resour. March, 1–12 (2021).
Google Scholar 
8.Klymus, K. E., Richter, C. A., Chapman, D. C. & Paukert, C. Quantification of eDNA shedding rates from invasive bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix. Biol. Conserv. 183, 77–84 (2015).Article 

Google Scholar 
9.Buxton, A. S., Groombridge, J. J., Zakaria, N. B. & Griffiths, R. A. Seasonal variation in environmental DNA in relation to population size and environmental factors. Sci. Rep. 7, 46294 (2017).ADS 
CAS 
Article 

Google Scholar 
10.Mächler, E., Deiner, K., Spahn, F. & Altermatt, F. Fishing in the water: Effect of sampled water volume on environmental DNA-based detection of macroinvertebrates. Environ. Sci. Technol. 50, 305–312 (2016).ADS 
Article 

Google Scholar 
11.Spens, J. et al. Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: Advantage of enclosed filter. Methods Ecol. Evol. 8, 635–645 (2016).Article 

Google Scholar 
12.Djurhuus, A. et al. Evaluation of filtration and DNA extraction methods for environmental DNA biodiversity assessments across multiple trophic levels. Front. Mar. Sci. 4, 314 (2017).Article 

Google Scholar 
13.Lugg, W. H., Griffiths, J., van Rooyen, A. R., Weeks, A. R. & Tingley, R. Optimal survey designs for environmental DNA sampling. Methods Ecol. Evol. 9, 1049–1059 (2017).14.Mauvisseau, Q. et al. Influence of accuracy, repeatability and detection probability in the reliability of species-specific eDNA based approaches. Sci. Rep. 9, 1–11 (2019).
Google Scholar 
15.Willoughby, J. R., Wijayawardena, B. K., Sundaram, M., Swihart, R. K. & DeWoody, J. A. The importance of including imperfect detection models in eDNA experimental design. Mol. Ecol. Resour. 16 , 837–844 (2016).16.Griffin, J. E., Matechou, E., Buxton, A. S., Bormpoudakis, D. & Griffiths, R. A. Modelling environmental DNA data; Bayesian variable selection accounting for false positive and false negative errors. J. R. Stat. Soc. Ser. C Appl. Stat. 69, 377–392 (2020).MathSciNet 
Article 

Google Scholar 
17.Lahoz-Monfort, J. J., Guillera-Arroita, G. & Tingley, R. Statistical approaches to account for false-positive errors in environmental DNA samples. Mol. Ecol. Resour. 16, 673–685 (2016).CAS 
Article 

Google Scholar 
18.Stratton, C., Sepulveda, A. J. & Hoegh, A. msocc: Fit and analyse computationally efficient multi-scale occupancy models in r. Methods Ecol. Evol. 11, 1113–1120 (2020).Article 

Google Scholar 
19.Tingley, R., Coleman, R., Gecse, N., van Rooyen, A. & Weeks, A. Accounting for false positive detections in occupancy studies based on environmental DNA: A case study of a threatened freshwater fish (Galaxiella pusilla). Environ. DNA 00, 1–10 (2020).
Google Scholar 
20.Schmidt, B. R., Kéry, M., Ursenbacher, S., Hyman, O. J. & Collins, J. P. Site occupancy models in the analysis of environmental DNA presence/absence surveys: A case study of an emerging amphibian pathogen. Methods Ecol. Evol. 4, 646–653 (2013).Article 

Google Scholar 
21.Vörös, J., Márton, O., Schmidt, B. R., Gál, J. T. & Jelić, D. Surveying Europe’s only cave-dwelling chordate species (Proteus anguinus) using environmental DNA. PLoS ONE 12, e0170945 (2017).Article 

Google Scholar 
22.Biggs, J. et al. Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus). Biol. Conserv. 183, 19–28 (2015).Article 

Google Scholar 
23.Cantera, I. et al. Optimizing environmental DNA sampling effort for fish inventories in tropical streams and rivers. Sci. Rep. 9, 3085 (2019).ADS 
Article 

Google Scholar 
24.Dejean, T. et al. Improved detection of an alien invasive species through environmental DNA barcoding: The example of the American bullfrog Lithobates catesbeianus. J. Appl. Ecol. 49, 953–959 (2012).Article 

Google Scholar 
25.Eiler, A., Löfgren, A., Hjerne, O., Nordén, S. & Saetre, P. Environmental DNA (eDNA) detects the pool frog (Pelophylax lessonae) at times when traditional monitoring methods are insensitive. Sci. Rep. 8, 5452 (2018).ADS 
Article 

Google Scholar 
26.Nakagawa, H. et al. Comparing local- and regional-scale estimations of the diversity of stream fish using eDNA metabarcoding and conventional observation methods. Freshw. Biol. 63, 569–580 (2018).CAS 
Article 

Google Scholar 
27.Royle, J. A. & Link, W. A. Generalized site occupancy models allowing for false positives and false negative errors. Ecology 87, 835–841 (2006).Article 

Google Scholar 
28.Mackenzie, D. I. & Kendall, W. L. How should detection probability be incorporated into estimates of relative abundance?. Ecology 83, 2387–2393 (2002).Article 

Google Scholar 
29.MacKenzie, D. D., Nichols, J. D., Hines, J. E., Knutson, M. G. & Franklin, A. B. Estimating site occupancy, colonization, and local extinction when a species is detected imperfectly. Ecology 84, 2200–2207 (2003).Article 

Google Scholar 
30.Tyre, A. J., Tenhumberg, B., Field, S. A., Niejalke, D. & Possingham, H. P. Improving precision and reducing bias in biological surveys: Estimating false-negative error rates. Ecol. Appl. 13, 1790–1801 (2003).Article 

Google Scholar 
31.Dorazio, R. M. & Erickson, R. A. EDNAOCCUPUANCY: An R package for multi-scale occupancy modeling of environmental DNA data. Mol. Ecol. Resour. 18, 368–380 (2018).CAS 
Article 

Google Scholar 
32.Guillera-Arroita, G., Lahoz-Monfort, J. J., van Rooyen, A. R., Weeks, A. R. & Tingley, R. Dealing with false positive and false negative errors about species occurrence at multiple levels. Methods Ecol. Evol. 8, 1081–1091 (2017).Article 

Google Scholar 
33.Cole, D. J. Parameter Redundancy and Identi Ability (CRC Press, Boca Raton, 2020).Book 

Google Scholar 
34.Diana, A., Matechou, E., Griffin, J. E., Buxtron, A. S. & Griffiths, R. A. An Rshiny app for modelling environmental DNA data: Accounting for false positve and false negative observation error. bioRxiv https://doi.org/10.1101/2020.12.09.417600 (2020).Article 

Google Scholar 
35.Biggs, J. et al. Analytical and methodological development for improved surveillance of the great crested newt. Defra Project WC1067. (2014).36.Sewell, D., Beebee, T. J. C. & Griffiths, R. A. Optimising biodiversity assessments by volunteers: The application of occupancy modelling to large-scale amphibian surveys. Biol. Conserv. 143, 2102–2110 (2010).Article 

Google Scholar 
37.Buxton, A. S., Tracey, H. & Downs, N. C. How reliable is the habitat suitability index as a predictor of great crested newt presence or absence?. Herpertological J. 31, 51–57 (2021).
Google Scholar 
38.R-Core Team. R: language and environment for statistical computing. (2020).39.Oldham, R. S., Keeble, J., Swan, M. J. S. & Jeffcote, M. Evaluating the suitability of habitat for the great crested newt (Triturus cristatus). Herpetol. J. 10, 143–155 (2000).
Google Scholar  More

1.Bryan, B. A. et al. China’s response to a national land-system sustainability emergency. Nature 559, 193–204 (2018).ADS 
CAS 
Article 

Google Scholar 
2.Zhang, W. J., Xue, X., Peng, F., You, Q. G. & Hao, A. H. Meta-analysis of the effects of grassland degradation on plant and soil properties in the alpine meadows of the Qinghai-Tibetan Plateau. Glob. Ecol. Conserv. 20, e00774 (2019).3.Pan, T., Zou, X. T., Liu, Y. J., Wu, S. H. & He, G. M. Contributions of climatic and non-climatic drivers to grassland variations on the Tibetan Plateau. Ecol. Eng. 108, 307–317 (2017).Article 

Google Scholar 
4.Shen, H. H., Wang, S. P. & Tang, Y. H. Grazing alters warming effects on leaf photosynthesis and respiration in Gentiana straminea, an alpine forb species. J. Plant. Ecol. 6, 418–427 (2013).Article 

Google Scholar 
5.Li, G. Y., Jiang, C. H., Cheng, T. & Bai, J. Grazing alters the phenology of alpine steppe by changing the surface physical environment on the northeast Qinghai-Tibet Plateau, China. J. Environ. Manage. 248, 109257 (2019).6.Li, Y. M. et al. Changes of soil microbial community under different degraded gradients of alpine meadow. Agric. Ecosyst. Environ. 222, 213–222 (2016).Article 

Google Scholar 
7.Guo, N. et al. Changes in vegetation parameters and soil nutrients along degradation and recovery successions on alpine grasslands of the Tibetan plateau. Agric. Ecosyst. Environ. 284, 106593 (2019).8.Lin, L. et al. Predicting parameters of degradation succession processes of Tibetan Kobresia grasslands. Solid Earth 6, 1237–1246 (2015).ADS 
Article 

Google Scholar 
9.Li, H. D. et al. Assessing revegetation effectiveness on an extremely degraded grassland, southern Qinghai-Tibetan Plateau, using terrestrial LiDAR and field data. Agric. Ecosyst. Environ. 282, 13–22 (2019).Article 

Google Scholar 
10.Wang, G. X., Qian, J., Cheng, G. D. & Lai, Y. M. Soil organic carbon pool of grassland soils on the Qinghai-Tibetan Plateau and its global implication. Sci. Total Environ. 291, 207–217. https://doi.org/10.1016/s0048-9697(01)01100-7 (2002).CAS 
Article 

Google Scholar 
11.Yuan, Z. Q. et al. Responses of soil organic carbon and nutrient stocks to human-induced grassland degradation in a Tibetan alpine meadow. CATENA 178, 40–48 (2019).CAS 
Article 

Google Scholar 
12.Askari, M. S. & Holden, N. M. Quantitative soil quality indexing of temperate arable management systems. Soil Till Res. 150, 57–67 (2015).Article 

Google Scholar 
13.Lima, A. C. R., Brussaard, L., Totola, M. R., Hoogmoed, W. B. & de Goede, R. G. M. A functional evaluation of three indicator sets for assessing soil quality. Appl. Soil Ecol. 64, 194–200 (2013).Article 

Google Scholar 
14.Masto, R. E., Chhonkar, P. K., Singh, D. & Patra, A. K. Alternative soil quality indices for evaluating the effect of intensive cropping, fertilisation and manuring for 31 years in the semi-arid soils of India. Environ. Monit. Assess 136, 419–435. https://doi.org/10.1007/s10661-007-9697-z (2008).CAS 
Article 
PubMed 

Google Scholar 
15.Zhou, H. et al. Changes in the soil microbial communities of alpine steppe at Qinghai-Tibetan Plateau under different degradation levels. Sci. Total Environ. 651, 2281–2291 (2019).ADS 
CAS 
Article 

Google Scholar 
16.Yang, C., Zhang, F. G., Liu, N., Hu, J. & Zhang, Y. J. Changes in soil bacterial communities in response to the fairy ring fungus Agaricus gennadii in the temperate steppes of China. Pedobiologia 69, 34–40 (2018).Article 

Google Scholar 
17.Li, J. J. & Yang, C. Inconsistent response of soil bacterial and fungal communities in aggregates to litter decomposition during short-term incubation. Peerj 7, e8078 (2019).18.Yang, C., Li, J. J., Liu, N. & Zhang, Y. J. Effects of fairy ring fungi on plants and soil in the alpine and temperate grasslands of China. Plant Soil 441, 499–510 (2019).CAS 
Article 

Google Scholar 
19.Yang, C., Liu, N. & Zhang, Y. J. Soil aggregates regulate the impact of soil bacterial and fungal communities on soil respiration. Geoderma 337, 444–452 (2019).ADS 
CAS 
Article 

Google Scholar 
20.Wardle, D. A. et al. Ecological linkages between aboveground and belowground biota. Science 304, 1629–1633 (2004).ADS 
CAS 
Article 

Google Scholar 
21.Wu, G.-L., Ren, G.-H., Dong, Q.-M., Shi, J.-J. & Wang, Y.-L. Above- and belowground response along degradation gradient in an alpine grassland of the Qinghai-Tibetan Plateau. Clean-Soil Air Water 42, 319–323. https://doi.org/10.1002/clen.201200084 (2014).CAS 
Article 

Google Scholar 
22.Che, R. X. et al. Degraded patch formation significantly changed microbial community composition in alpine meadow soils. Soil Till Res. 195, 104426 (2019).23.Aßhauer, K. P., Wemheuer, B., Daniel, R. & Meinicke, P. Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data. Bioinformatics 31, 2882–2884 (2015).Article 

Google Scholar 
24.Harris, R. B. Rangeland degradation on the Qinghai-Tibetan plateau: a review of the evidence of its magnitude and causes. J. Arid Environ. 74, 1–12. https://doi.org/10.1016/j.jaridenv.2009.06.014 (2010).ADS 
CAS 
Article 

Google Scholar 
25.Ren, G., Shang, Z., Long, R., Hou, Y. & Deng, B. The relationship of vegetation and soil differentiation during the formation of black-soil-type degraded meadows in the headwater of the Qinghai-Tibetan Plateau China. Environ. Earth Sci. 69, 235–245. https://doi.org/10.1007/s12665-012-1951-1 (2013).Article 

Google Scholar 
26.Zhang, Y. et al. Diversity of nitrogen-fixing, ammonia-oxidizing, and denitrifying bacteria in biological soil crusts of a revegetation area in Horqin Sandy Land Northeast China. Ecol. Eng. 71, 71–79. https://doi.org/10.1016/j.ecoleng.2014.07.032 (2014).Article 

Google Scholar 
27.Wang, Y. et al. Effects of grassland degradation on ecological stoichiometry of soil ecosystems on the Qinghai-Tibet Plateau. Sci. Total Environ. 722, 137910. https://doi.org/10.1016/j.scitotenv.2020.137910 (2020).ADS 
CAS 
Article 
PubMed 

Google Scholar 
28.Zhang, Y. et al. Soil bacterial and fungal diversity differently correlated with soil biochemistry in alpine grassland ecosystems in response to environmental changes. Sci. Rep. 7, 43077. https://doi.org/10.1038/srep43077 (2017).ADS 
Article 
PubMed 
PubMed Central 

Google Scholar 
29.Hartmann, M. et al. Resistance and resilience of the forest soil microbiome to logging-associated compaction. ISME J. 8, 226–244. https://doi.org/10.1038/ismej.2013.141 (2014).CAS 
Article 
PubMed 

Google Scholar 
30.Liu, S. B., Zamanian, K., Schleuss, P. M., Zarebanadkouki, M. & Kuzyakov, Y. Degradation of tibetan grasslands: consequences for carbon and nutrient cycles. Agric. Ecosyst. Environ. 252, 93–104 (2018).CAS 
Article 

Google Scholar 
31.He, S. Y. & Richards, K. Impact of meadow degradation on soil water status and pasture managementA case study in tibet. Land Degrad. Dev. 26, 468–479. https://doi.org/10.1002/ldr.2358 (2015).Article 

Google Scholar 
32.Yergeau, E., Hogues, H., Whyte, L. G. & Greer, C. W. The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses. ISME J. 4, 1206–1214. https://doi.org/10.1038/ismej.2010.41 (2010).CAS 
Article 
PubMed 

Google Scholar 
33.Eichorst, S. A. et al. Genomic insights into the Acidobacteria reveal strategies for their success in terrestrial environments. Environ. Microbiol. 20, 1041–1063 (2018).CAS 
Article 

Google Scholar 
34.Fang, D. X. et al. Microbial community structures and functions of wastewater treatment systems in plateau and cold regions. Bioresour. Technol. 249, 684–693 (2018).CAS 
Article 

Google Scholar 
35.Mukhopadhya, I., Hansen, R., El-Omar, E. M. & Hold, G. L. IBD—what role do proteobacteria play?. Nat. Rev. Gastroenterol. Hepatol. 9, 219–230. https://doi.org/10.1038/nrgastro.2012.14 (2012).CAS 
Article 
PubMed 

Google Scholar 
36.Kjoller, A. H. & Struwe, S. Fungal communities, succession, enzymes, and decomposition (2002).37.Poll, C., Brune, T., Begerow, D. & Kandeler, E. Small-scale diversity and succession of fungi in the detritusphere of rye residues. Microbial. Ecol. 59, 130–140. https://doi.org/10.1007/s00248-009-9541-9 (2010).Article 

Google Scholar 
38.Jangid, K. et al. Land-use history has a stronger impact on soil microbial community composition than aboveground vegetation and soil properties. Soil Biol. Biochem. 43, 2184–2193. https://doi.org/10.1016/j.soilbio.2011.06.022 (2011).CAS 
Article 

Google Scholar 
39.Cao, C. et al. Soil bacterial community responses to revegetation of moving sand dune in semi-arid grassland. Appl. Microbiol. Biotechnol. 101, 6217–6228. https://doi.org/10.1007/s00253-017-8336-z (2017).CAS 
Article 
PubMed 

Google Scholar 
40.Tripathi, B. M. et al. Tropical soil bacterial communities in Malaysia: pH dominates in the equatorial tropics too. Microbial. Ecol. 64, 474–484. https://doi.org/10.1007/s00248-012-0028-8 (2012).Article 

Google Scholar 
41.Chu, H. et al. Bacterial community dissimilarity between the surface and subsurface soils equals horizontal differences over several kilometers in the western Tibetan Plateau. Environ. Microbiol. 18, 1523–1533. https://doi.org/10.1111/1462-2920.13236 (2016).CAS 
Article 
PubMed 

Google Scholar 
42.Wu, X. et al. Bacterial communities in the upper soil layers in the permafrost regions on the Qinghai-Tibetan plateau. Appl. Soil Ecol. 120, 81–88. https://doi.org/10.1016/j.apsoil.2017.08.001 (2017).Article 

Google Scholar 
43.Yang, C. et al. Assessing the effect of soil salinization on soil microbial respiration and diversities under incubation conditions. Appl. Soil Ecol. https://doi.org/10.1016/j.apsoil.2020.103671 (2020).Article 

Google Scholar 
44.Langille, M. G. I. et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat. Biotechnol. 31, 814. https://doi.org/10.1038/nbt.2676 (2013).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
45.Mermin, J. et al. Reptiles, amphibians, and human Salmonella infection: a population-based, case-control study. Clin. Infect. Dis. 38, S253–S261. https://doi.org/10.1086/381594 (2004).Article 
PubMed 

Google Scholar 
46.Wang, J. et al. Plant community ecological strategy assembly response to yak grazing in an alpine meadow on the eastern Tibetan Plateau. Land Degrad. Dev. 29, 2920–2931. https://doi.org/10.1002/ldr.3050 (2018).Article 

Google Scholar 
47.Ji, S., Geng, Y., Li, D. & Wang, G. Plant coverage is more important than species richness in enhancing aboveground biomass in a premature grassland, northern China. Agric. Ecosyst. Environ. 129, 491–496. https://doi.org/10.1016/j.agee.2008.11.002 (2009).Article 

Google Scholar 
48.Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336. https://doi.org/10.1038/nmeth.f.303 (2010).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
49.Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 6, 1621–1624. https://doi.org/10.1038/ismej.2012.8 (2012).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
50.Chen, W. et al. Consistent responses of surface- and subsurface soil fungal diversity to N enrichment are mediated differently by acidification and plant community in a semi-arid grassland. Soil Biol. Biochem. 127, 110–119. https://doi.org/10.1016/j.soilbio.2018.09.020 (2018).CAS 
Article 

Google Scholar 
51.Kanehisa, M. et al. KEGG for linking genomes to life and the environment. Nucleic Acids Res. 36, D480–D484. https://doi.org/10.1093/nar/gkm882 (2008).CAS 
Article 
PubMed 

Google Scholar  More

The development of massive sequencing has provided a relatively inexpensive method to obtain the transcriptome of a species. Taking advantage of this technique, we used a previously obtained transcriptome of P. acuta to identify 18 genes related to different pathways of interest in ecotoxicology and then examined how exposure to phthalates changed the transcription of these genes. The processes of interest include DNA repair, the stress response, detoxification, apoptosis, immunity, energy reserves, and lipid transportation. There is a growing interest in combining ecologically relevant endpoints with biochemical and molecular parameters to seek a more integrative analysis. In this sense, increasing the number of described genes will allow for the design of standard arrays that could be used in combination with toxicity tests. In this way, initiatives such as the Adverse Outcome Pathway wiki24 will increase its relevance in assessing old and new compounds and provide putative mechanisms of action to explain the differences to the animals’ specific physiology. Furthermore, increasing knowledge at the molecular level in P. acuta supports its use as a representative of freshwater gastropods in toxicity analysis. There is a lack of model freshwater mollusks, which is one of the animal groups whose pollution response is currently less known.The 18 newly identified genes evaluated in this work show homology with those previously described in other species, as expected, mainly with the freshwater snail Biomphalaria glabrata, which belongs to the Planorbidae family. rad21 and rad50 are both involved in DNA repair: rad21 is an essential gene encoding a DNA double-strand break repair protein21, and rad50 is a member of the protein complex MRN (including Mre11, RAD50, and Nbs1) that functions in DNA double-strand break repair to recognize and process DNA ends as well as a signal for cell cycle arrest25. There is very little information about these genes in mollusks, with only one report in Crassostrea gigas for rad5026. The relevance of these genes is that their detection can be combined with other methodologies, such as the comet assay, to perform an integrated study to determine whether a compound is genotoxic and whether the organism has the ability to compensate for the damage.The Cat and SOD Mn genes allow us to evaluate the status of oxidative stress. Oxidative stress analysis is usually focused on biochemical parameters, such as enzyme activity. However, it should also include a transcriptional activity study because it can provide additional information about the mid- and long-term responses. Protein turnover can also be relevant in the response, especially in chronic exposure to toxicants. Detoxification mechanisms are also important to assess the response to toxicants. GST activity is one of the most used methods to assess detoxification27, but it does not differentiate between the members involved. The situation is similar regarding cytochrome P450s, which show high diversity with many roles in the cell28. Our identification of the Cyp72a15 gene increases the number of cytochromes 450 s described in P. acuta. Evaluating changes in these genes can help to elucidate how the organism can process the toxicants.The sHSP17.9 and HSC70-4 genes extend the battery of genes available to assess the stress response of P. acuta. sHSP17.9 is difficult to match with other species’ genes because while they all have an alpha-crystallin domain, there is no other sequence that presently allows for homology to be established. Additional functional studies will help to search for homology. It is worth mentioning that HIF1α offers a new aspect of stress related to hypoxia29. The stress response mainly focuses on the canonical heat shock proteins, so other mechanisms involved in specific stresses, such as hypoxia, are usually neglected. With the identification of HIF1a in P. acuta, researchers can evaluate the effect of a toxicant on oxygen intake in this species.The remaining identified genes allow for the analysis of pathways that can also be altered by toxicants, like apoptosis (AIF3), the immune system (ApA), energy reserves (PYGL), and lipid transport (ORP8). To our knowledge, in this study these genes have been analyzed for the first time concerning pollution in freshwater mollusks. The last three genes, DNMT1, KATB6, and HDAC1, are involved in epigenetic mechanisms. There is increasing evidence that epigenetic regulation is one of the long-term effects of toxicants. However, the genes involved in this process in invertebrates are still poorly represented in toxicity analysis. The description of these three genes opens the possibility of analyzing their role in the epigenetic response and its relevance in the transgenerational effects that have started to be described with different toxicants30,31,32.Plastics in the environment are a growing problem. During the degradation process, the polymers themselves and the compounds used as additives, including phthalates, are released. Hence, the presence of phthalates is increasing in the environment5,33,34. We analyzed three phthalates in this work, namely BBP, DEP, and DEHP; they showed a differential impact in P. acuta. DEP and DEHP, did not alter any of the mRNA levels. Researchers have described previously that both phthalates can alter the physiology of invertebrates16,35,36,37,38, including mollusks39,40,41. Other phthalates can also alter development and growth, which could be related to the endocrine-disrupting activity described for those chemicals. The molecular mechanisms involved are still under investigation, but some data are available. In the clam Venerupis philippinarum, DEHP alters the immune response40. In H. diversicolor, DBP affects oxidative stress, lipid and energy metabolism, and osmoregulation17. In other invertebrates, including Chironomus riparius42, Drosophila melanogaster43, and Caenorhabditis elegans15, phthalates alter endocrine pathways. The changes affect the ecdysone response as well as the expression of insulin-like peptide. Other pathways are also affected by phthalates, such as oxidative stress and detoxification routes44 and the stress response14. Finally, in C. elegans, exposure to environmentally relevant concentrations of diethylhexyl phthalate produces genomic instability by altering the expression of genes involved in DNA repair during meiosis37. It is clear then that phthalates can have a broad spectrum of actions in the cell, with a significant alteration of metabolism but primarily affecting oxidative stress and the endocrine system.The previous studies performed in mollusks have revealed alterations in several physiological processes; the analyzed molecular mechanisms mainly involved oxidative stress and immunity17,41. A recent review of the impact of phthalates on aquatic animals summarizes the effects observed, suggesting that activation of the detoxification system (cytochrome P450s) and endocrine system receptors of aquatic animals cause oxidative stress, metabolic disorders, endocrine disorders, and immunosuppression8. It would activate a cascade response that could cause genotoxicity and cell apoptosis, resulting in the disruption of growth and development. Considering this, the absence of a response observed in P. acuta exposed to DEP and DEHP is striking. The differences observed can be assigned to the type of analysis (molecular vs. physiological), the exposure time (1 week vs. a few hours or days), the concentration used (μg/L vs. mg/L), and evidently, the species used. Additional research will help elucidate the differential response in P. acuta compared with other organisms. However, it is essential to highlight that the obtained results suggest that P. acuta can manage the environmentally relevant doses of DEP and DEHP used in this work. This species may be less sensitive to these phthalates, but this eventually will require further research, including the use of other methodological approaches, to confirm it.In contrast to DEP and DEHP, BBP showed a marked effect: it increased the mRNA levels of almost all the analyzed genes. It is essential to consider that most studies on invertebrates that involve transcriptional activity analysis use arthropods and short exposure times14,44,45,46. Limited data are available on mollusks and, usually, they are marine representatives40,47. To our knowledge, this is the first study on a freshwater snail that shows that BBP can produce a substantial effect on cell metabolism. Several of the altered pathways can explain, in some way, the effects observed in other organisms, like DNA repair by the alteration of rad21 and rad50, which are related to DNA damage, or the alteration of the genes involved in histone and DNA modification (KAT6B, HDAC1, and DNMT1), which are related to epigenetic regulation. Apoptosis, which phthalates can also alter, also seems to be modulated in P. acuta by altering the AIF3 and the casp3 genes. Furthermore, the three phases of the detoxification could be acting since the genes tested (three cytochrome P450s, three GSTs, and MRP-1) were upregulated.Genes involved in oxidative stress and the stress response were also altered, as shown by the changes in the mRNA levels of Cat, SODs, stress proteins, and the hypoxia-related transcription factor genes. These changes support the alteration of oxidative stress, the stress response, and detoxification, backing previous analysis and adding new insight about the mechanisms involved in modulating these processes. In this sense, the absence of changes in GSTm1 supports a differential role for each GST family member in the response to toxicants. The altered acetylcholinesterase mRNA level also suggests effects in the nervous system, requiring additional research to elucidate the damage to the central nervous system. Finally, the alteration of PYGL, ApA, and ORP8, involved in energy metabolism, immunity, and lipid transport, respectively, shows that P. acuta responds to BBP in a way that has been observed in other organisms. In summary, the present gene profile obtained in response to BBP in P. acuta supports the proposed mechanisms and cellular processes in studies with other animals8. Immunity, oxidative stress, the stress response, detoxification, apoptosis, epigenetic modulation, DNA repair, lipid metabolism, and energy metabolism are modulated. The nervous system could also be affected. Of note, some genes showed differences in transcription based on the phthalate concentration. These findings suggest there are subtle differences, and additional kinetic analysis is required to elucidate early and late activated genes and the relevance of the damage for the population’s future.The obtained results are in line with previous studies in other organisms, which have confirmed that BBP can induce different types of damage such as apoptosis48, genotoxicity49, oxidative stress50, stress response activation45, or endocrine disruption14. Although there are studies in invertebrates showing the impact on development and other physiological processes39,51, most of them did not focus on the putative mode of action, with only a few of them trying to delve into the response mechanisms. Here we have shown that BBP can extensively affect the cell transcriptional activity in P. acuta. These results could be considered to reflect specific alterations on these pathways. This scenario would mean that BBP is the most active phthalate in P. acuta, with a broad spectrum of action and a potential effect on many pathways. However, the more probable picture is something that has been recently proposed: alterations in the oxidative stress response and the endocrine system cause a cascade of responses that affect different pathways and ultimately block growth and development8. It is relevant to keep in mind that BBP is a known endocrine disruptor47. A recent study in Daphnia magna provides some insight. Specifically, RNA-Seq revealed that genes involved in signal transduction, cell communication, and embryonic development were significantly down-regulated, while those related to biosynthesis, metabolism, cell homeostasis, and redox homeostasis were remarkably upregulated upon BBP exposure46. Although the organism and the stage analyzed are different from our study, those results support the idea that BBP can simultaneously alter multiple pathways, and it fits better with the regulatory role of the endocrine system and the extensive affection by oxidative stress.As stated before, the results obtained in this work show that DEP and DEHP had no apparent effect to P. acuta after 1 week exposure to environmentally relevant concentrations. However, BBP showed a strong effect. The difference in response could be due to several reasons that need to be explored in future work. One possibility is the structure of each compound. In this sense, BBP has two benzene rings while DEP and DEHP have only one. This factor could determine the biological activity of these compounds. Another possibility is that DEP and DEHP have effects earlier than the time studied, and the cell returned to the basal state, being able to process and remove the compounds. Finally, it cannot be dismissed that DEP and DEHP are not toxic to P. acuta, at least at environmentally relevant concentrations. In any case, BBP alters the metabolism of this species and produces a broad impact on different pathways. Additional research should be done in P. acuta and other freshwater species to determine the impact on organisms based on the freshwater ecosystem food web. More

Volcanic ash soilWe used commercially available Kanuma soil as volcanic ash soil (fine-grained pumice, Akagi Engei Co., Ltd.) for growth experiment 1. Kanuma soil is a fully weathered pyroclastic fall from the eruption of Mt. Akagi 44,000 years ago19. The soil contains 30.8% aluminum (allophane and imogolite) and 1.4% iron (ferrihydrite)20. For growth experiment 2, we used three natural volcanic ash soil types—immature soil of pumice (Kanuma soil, C horizon), as well as mature soils of andosol (A–B horizon) and topsoil (the surface of andosol, P to A horizon)—collected from a riverbed in Kanuma City (36°35′ N, 139°44′ E; 200 m a.s.l.), central Japan, where the vegetation is a cypress forest. This place is managed by the Kanuma Civil Engineering Office. The topsoil was collected at a depth of approximately 0–10 cm from the soil surface after removing the fallen leaves on the soil surface. The andosol layer, typically distributed at a depth of approximately 10–75 cm, was collected from a depth of approximately 10–30 cm. Below the andosol layer, the Akadama soil layer is distributed; further below, the pumice Kanuma soil is distributed. The pumice was collected approximately 50 cm under the Akadama layer.Temporal information on soil formation was confirmed by direct radiocarbon dating of the soil samples. After removing soil carbonate with 1.0 M HCl, the total organic fraction was analyzed using an accelerator mass spectrometer (0.5MV compact AMS system, NEC) at the laboratory of radiocarbon dating, University of Tokyo. Conventional radiocarbon age after correction of isotopic fractionation with δ13C values was calibrated to a calendar date with the calibration dataset IntCal1321.The elemental analysis of total phosphorus, nitrogen, and carbon in the soil samples was performed by Createrra Inc. (http://www.createrra.co.jp/english/top.html).Plant speciesOn the volcanic ash soil of Mt. Fuji, Japan’s highest volcano, vegetation in primary succession generally changes from herbaceous plants such as Fallopia japonica (Houtt.) Ronse Decr. var. japonica to nitrogen-fixing alder plants, and finally to non-nitrogen fixing Betula ermanii Cham22,23. Hence, we used three species—F. japonica, the alder species Alnus inokumae Murai et Kusaka, and B. ermanii—owned by and grown in our research institute, Nikko Botanical Garden, for the growth experiments. Experimental research on these plants, including the collection of plant material, comply with the relevant institutional, national, and international guidelines and legislation.Litter incubation experimentSamples (1 g) of F. japonica litter leaves—collected upon leaf fall on an autumn day, dried at 80 °C for at least 48 h, and then crushed—were placed in cultivating tubes (n = 5). Then, 5 g of wet soil from the Nikko Botanical Garden (36°45′ N, 139°35′ E; 647 m a.s.l.) in Nikko, central Japan, was added to 500 mL of water and stirred (solution I). As inoculation, 0.1 mL of the supernatant of solution I was added to the tubes24. Considering that the amounts of phosphorus and nitrogen in the solution I were approximately 0.003 mg/L and 0.3 mg/L, respectively, they were determined to have not affected the initial value (t = 0). Next, 2 mL of water was added to the tubes, which were then kept at 30 °C. The tubes were left open to maintain an aerobic environment. The efflux of phosphorus and nitrogen from the leaves was measured every week for ten weeks. For these measurements, 5 mL of water was added and the tube was centrifuged for 10 min (solution II). The supernatant of solution II was then used for phosphorus and nitrogen measurements, and the residue was continuously kept at 30 °C.Growth experimentsGrowth experiments were conducted in an open-type greenhouse in Nikko Botanical Garden. The greenhouse is only vinyl on the ceiling and good ventilation to keep the temperature constant. The mean monthly highest and lowest temperatures and the monthly precipitation observed in the botanical garden during the cultivation period are provided in Table 1. In the growth experiments, irrigation with tap water was provided to the plants and litter leaves in the morning and evening. The phosphorus and nitrogen concentration of the tap water were approximately 0.03 mg/L and 0.25 mg/L respectively.Table 1 Nikko botanical garden weather data (May–October 2019).Full size tableGrowth experiment 1: Comparative experiment on the growth of plant species with and without litterThe seedlings used for the experiment were from the species F. japonica, A. inokumae, and B. ermanii. A similar seedling size was used for each plant species. Seedlings of A. inokumae coexist with N-fixing actinomycetes.Six plants per species were collected before cultivation (t = 0) and dried in an oven at 80 °C for at least 48 h to measure the dry weight. There were four experimental groups for each species: a control (Con), a nitrogen addition (N: 10 mM NH4NO3), a phosphorus addition (P: 10 mM NaH2PO4), and a nitrogen and phosphorus addition (NP: 10 mM NH4NO3 + 10 mM NaH2PO4). Once a week, 50 mL of each nutrients was added to a 0.25-L garden pot. To verify whether the addition of litter (denoted by +) improved plant growth, litter leaves of F. japonica were placed on the soils. To verify if nutrients leached from litter sustained plant growth, we also combined nutrient and litter additions (Con+, N+, P+, NP+). When nutrients were added to the soil once a week, litter bag was removed before fertilizer application and returned after that.To reproduce how litter is deposited and supplies nutrients on volcanic ash soil in primary succession, F. japonica litter was collected in Nikko in the autumn of 2018 and dried at 80 °C or 2 days or more (the same litter was used in incubation). Approximately 9 g of litter leaves was packed in a tea mesh bag25 to prevent it from flying in the wind and placed on the soil surface of the garden pots. As indicated by the equation below, the amount of litter added to the 8 × 8 cm (0.0064 m2) garden pot used in this experiment amounts to approximately three years of litter production when converted to the amount of leaf litter in a 15-year-old alder forest, i.e., about 430 g/m2 per year26.$$frac{9,g}{{430frac{g}{{ m^{2} }} yr times 0.0064 m^{2} }} cong 3.3 yr$$Six seedlings per group of A. inokumae and B. ermanii were cultivated for approximately 2 months (June 7–August 22, 2019) and 12 seedlings per group of F. japonica were cultivated for about 1 month (September 10–October 15, 2019). The experiment was stopped after 1 month for F. japonica as it grew rapidly in 2 nutrient conditions (NP, NP+) and the roots overflowed from the garden pot. At the end of the experiment, growth was evaluated by measuring dry weight after drying seedlings at 80 °C for at least 48 h. Subsequently, the total phosphorus and nitrogen content of the dried seedlings were also measured (chemical analysis).The mass of phosphorus leached from litter during the cultivation period was calculated from the difference in the phosphorus contents of the litter before and after cultivation.Growth experiment 2: Comparative experiment on plant growth with old organic matterEight F. japonica seedlings were cultivated in three different soil-types (pumice, andosol, and topsoil, as mentioned above) under three experimental conditions (Con, N, P, same nutrition as growth experiment 1) from May 29 to July 12, 2019. These plants were then harvested and oven-dried at 80 °C for at least 48 h to measure dry weight. Subsequently, the total phosphorus and nitrogen content of the seedlings were also measured (chemical analysis).Chemical analysisPhosphorusWe used the dry destruction method to pretreat total phosphorus measurements in plant tissue27. A sample of the plant (0.05 g) was burned at 550 °C for 1 h. The plant ash was dissolved in 10 mL of 2 M H2SO4 and shaken for over 16 h; then, the solution was filtered. The filtrate was diluted at a 1:10 ratio with tris(hydroxymethyl)aminomethane (pH 8.0).The soil for available phosphorus were pretreated by Truog’ s method28. The soil (0.05 g) was dissolved in 10 mL of 0.002 M H2SO4, shaken for 30 min, and the solution was filtered. The filtrate was diluted at a 1:10 ratio with water.The amount of phosphorus in the sample solution was measured by the molybdenum blue colorimetric method29.NitrogenThe total nitrogen in plant tissue was measured using an elemental analyzer (EA; Vario Macro cube, Elementar, Germany). A few milligrams of the dried plant sample were placed in a tin capsule for EA combustion. EA carried out sample combustion and N2 separation/detection from the combusted gases and provided us with nitrogen contents.The soil sample preparation for available nitrogen measurements was based on the incubation methodology30. Half of the sampled soils were analyzed fresh, and the other half incubated for four weeks at 30 °C before analysis. 2 M KCl (20 mL) was added to 2 g of the soil sample; the solution was shaken for 1 h and filtered. The filtrate was collected, and the volume of nitrogen was measured by indophenol blue absorptiometry after reducing all to ammonia using Pack Test WAK-TNi (Kyoritsu Chemical-Check Lab., Corp, Tokyo, Japan). Available nitrogen was taken as the difference in the concentration of inorganic nitrogen (NO3-N, NO2-N and NH4-N) between incubated and fresh soil.Statistical analysisAll statistical analyses were performed with EZR31 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics. The figure’s values are mean ± SE. Intergroup differences for nutrition conditions in soil, and soil-types were evaluated using non-parametric Kruskal–Wallis with post-hoc Steel–Dwass tests. In addition, comparisons between with or without litter were evaluated using two-tailed Mann–Whitney U-test. p values are * p  More

1.Frölicher, T. L., Fischer, E. M. & Gruber, N. Marine heatwaves under global warming. Nature 560, 360–364 (2018).ADS 
PubMed 
Article 
CAS 
PubMed Central 

Google Scholar 
2.Oliver, E. C. J. et al. Longer and more frequent marine heatwaves over the past century. Nat. Commun. 9, 1–12 (2018).CAS 
Article 

Google Scholar 
3.Smale, D. A. et al. Marine heatwaves threaten global biodiversity and the provision of ecosystem services. Nat. Clim. Change 9, 306–312 (2019).ADS 
Article 

Google Scholar 
4.Hobday, A. J. et al. A hierarchical approach to defining marine heatwaves. Prog. Oceanogr. 141, 227–238 (2016).ADS 
Article 

Google Scholar 
5.Dee, L. E. E. et al. Temperature variability alters the stability and thresholds for collapse of interacting species. Philos. Trans. R. Soc. Biol. Sci. 375, 20190457 (2020).Article 

Google Scholar 
6.Leung, J. Y. S., Russell, B. D. & Connell, S. D. Adaptive responses of marine gastropods to heatwaves. One Earth 1, 374–381 (2019).Article 

Google Scholar 
7.Whiteley, N. M. & Mackenzie, C. L. Physiological responses of marine invertebrates to thermal stress. in Stressors in the Marine Environment (eds. Solan, M. & Whiteley, N. M.) 56–72 (Oxford University Press, 2016). https://doi.org/10.1093/acprof:oso/9780198718826.003.0004.8.Lonhart, S. I., Jeppesen, R., Beas-luna, R., Crooks, J. A. & Lorda, J. Shifts in the distribution and abundance of coastal marine species along the eastern Pacific Ocean during marine heatwaves from 2013 to 2018. Mar. Biodivers. Rec. 8, 1–15 (2019).
Google Scholar 
9.Smale, D. A. & Wernberg, T. Extreme climatic event drives range contraction of a habitat-forming species. Proc. R. Soc. B Biol. Sci. 280, 20122829 (2013).Article 

Google Scholar 
10.Garrabou, J. et al. Mass mortality in Northwestern Mediterranean rocky benthic communities: Effects of the 2003 heat wave. Glob. Change Biol. 15, 1090–1103 (2009).ADS 
Article 

Google Scholar 
11.Frölicher, T. L. & Laufkötter, C. Emerging risks from marine heat waves. Nat. Commun. 9, 2015–2018 (2018).Article 
CAS 

Google Scholar 
12.Cheung, W. W. L. & Frölicher, T. L. Marine heatwaves exacerbate climate change impacts for fisheries in the northeast Pacific. Sci. Rep. 10, 1–10. https://doi.org/10.1038/s41598-020-63650-z (2020).CAS 
Article 

Google Scholar 
13.Caputi, N. et al. Management adaptation of invertebrate fisheries to an extreme marine heat wave event at a global warming hot spot. Ecol. Evolut. https://doi.org/10.1002/ece3.2137 (2016).Article 

Google Scholar 
14.Verdelhos, T., Marques, J. C. & Anastácio, P. Behavioral and mortality responses of the bivalves Scrobicularia plana and Cerastoderma edule to temperature, as indicator of climate change’s potential impacts. Ecol. Indic. 58, 95–103 (2015).Article 

Google Scholar 
15.Shanks, A. L. et al. Marine heat waves, climate change, and failed spawning by coastal invertebrates. Limnol. Oceanogr. 65, 627–636 (2020).ADS 
Article 

Google Scholar 
16.Morgan, E. A., Brown, A., Ciotti, B. J. & Panton, A. Effects of temperature stress on ecological processes. in Stressors in the Marine Environment (eds. Solan, M. & Whiteley, N. M.) 213–227 (Oxford University Press, 2016). https://doi.org/10.1093/acprof:oso/9780198718826.003.0012.17.Beukema, J. J. & Dekker, R. Winters not too cold, summers not too warm: long-term effects of climate change on the dynamics of a dominant species in the Wadden Sea: the cockle Cerastoderma edule L. Mar. Biol. 167, 1–8 (2020).Article 

Google Scholar 
18.Sousa, R. et al. Die-offs of the endangered pearl mussel Margaritifera margaritifera during an extreme drought. Aquat. Conserv. Mar. Freshw. Ecosyst. 28, 1244–1248 (2018).Article 

Google Scholar 
19.Smale, D. A., Yunnie, A. L. E., Vance, T. & Widdicombe, S. Disentangling the impacts of heat wave magnitude, duration and timing on the structure and diversity of sessile marine assemblages. PeerJ 2015, 1–23 (2015).
Google Scholar 
20.McLusky, D. S. & Elliott, M. The Estuarine Ecosystem: Ecology (Threats and Management. Oxford Press, 2004).Book 

Google Scholar 
21.Johnson, R. G. Temperature variation in the infaunal environment of a sand flat. Limnol. Oceanogr. 10, 114–120 (1965).ADS 
Article 

Google Scholar 
22.Amorim, V. E. et al. Immunological and oxidative stress responses of the bivalve Scrobicularia plana to distinct patterns of heatwaves. Fish Shellfish Immunol. 106, 1067–1077 (2020).CAS 
PubMed 
Article 

Google Scholar 
23.Grilo, T. F. F., Cardoso, P. G. G., Dolbeth, M., Bordalo, M. D. D. & Pardal, M. Â. A. Effects of extreme climate events on the macrobenthic communities’ structure and functioning of a temperate estuary. Mar. Pollut. Bull. 62, 303–311 (2011).CAS 
PubMed 
Article 

Google Scholar 
24.Dolbeth, M. et al. Long-term changes in the production by estuarine macrobenthos affected by multiple stressors. Estuar. Coast. Shelf Sci. 92, 10–18 (2011).ADS 
Article 

Google Scholar 
25.Ouellette, D. et al. Effects of temperature on in vitro sediment reworking processes by a gallery biodiffusor, the polychaete Neanthes virens. Mar. Ecol. Prog. Ser. 266, 185–193 (2004).ADS 
Article 

Google Scholar 
26.Nagelkerken, I. & Munday, P. L. Animal behaviour shapes the ecological effects of ocean acidification and warming: Moving from individual to community-level responses. Glob. Chang. Biol. 22, 974–989 (2016).ADS 
PubMed 
Article 

Google Scholar 
27.Solan, M., Bennett, E. M., Mumby, P. J., Leyland, J. & Godbold, J. A. Benthic-based contributions to climate change mitigation and adaptation. Philos. Trans. R. Soc. B Biol. Sci. 375, 20190107 (2020).Article 

Google Scholar 
28.Kristensen, E. & Kostka, J. E. Macrofaunal burrows and irrigation in marine sediment: Microbiological and biogeochemical interactions. in Interactions Between Macro‐ and Microorganisms in Marine Sediments (eds. Kristensen, E., Haese, R. R. & Kostka, J. E.), 125–157 (American Geophysical Union, 2013). https://doi.org/10.1029/CE060p0125.29.Cozzoli, F. et al. Biological and physical drivers of bio-mediated sediment resuspension: A flume study on Cerastoderma edule. Estuar. Coast. Shelf Sci. 241, 106824 (2020).Article 

Google Scholar 
30.Soissons, L. M. et al. Sandification vs. muddification of tidal flats by benthic organisms: A flume study. Estuar. Coast. Shelf Sci. 228, 106355 (2019).Article 

Google Scholar 
31.Fernandes, S., Sobral, P. & Costa, M. H. Nereis diversicolor effect on the stability of cohesive intertidal sediments. Aquat. Ecol. 40, 567–579 (2006).CAS 
Article 

Google Scholar 
32.Paramor, O. A. L. & Hughes, R. G. The effects of bioturbation and herbivory by the polychaete Nereis diversicolor on loss of saltmarsh in south-east England. J. Appl. Ecol. 41, 449–463 (2004).Article 

Google Scholar 
33.Dolbeth, M., Crespo, D., Leston, S. & Solan, M. Realistic scenarios of environmental disturbance lead to functionally important changes in benthic species-environment interactions. Mar. Environ. Res. 150, 104770 (2019).CAS 
PubMed 
Article 

Google Scholar 
34.Godbold, J. A. & Solan, M. Long-term effects of warming and ocean acidification are modified by seasonal variation in species responses and environmental conditions. Philos. Trans. R. Soc. B Biol. Sci. 368, 20130186 (2013).Article 
CAS 

Google Scholar 
35.Godbold, J. A., Hale, R., Wood, C. L. & Solan, M. Vulnerability of macronutrients to the concurrent effects of enhanced temperature and atmospheric pCO2 in representative shelf sea sediment habitats. Biogeochemistry 135, 89–102 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
36.Sorte, C. J. B., Fuller, A. & Bracken, M. E. S. Impacts of a simulated heat wave on composition of a marine community. Oikos 119, 1909–1918 (2010).Article 

Google Scholar 
37.Pansch, C. et al. Heat waves and their significance for a temperate benthic community: A near-natural experimental approach. Glob. Change Biol. 24, 4357–4367 (2018).ADS 
Article 

Google Scholar 
38.Queirós, A. M. et al. A bioturbation classification of European marine infaunal invertebrates. Ecol. Evol. 3, 3958–3985 (2013).PubMed 
PubMed Central 
Article 

Google Scholar 
39.Wrede, A., Beermann, J., Dannheim, J., Gutow, L. & Brey, T. Organism functional traits and ecosystem supporting services—A novel approach to predict bioirrigation. Ecol. Indic. 91, 737–743 (2018).Article 

Google Scholar 
40.Crespo, D. et al. New climatic targets against global warming: Will the maximum 2 °C temperature rise affect estuarine benthic communities. Sci. Rep. 7, 1–14 (2017).Article 
CAS 

Google Scholar 
41.Galasso, H. L., Richard, M., Lefebvre, S., Aliaume, C. & Callier, M. D. Body size and temperature effects on standard metabolic rate for determining metabolic scope for activity of the polychaete Hediste (Nereis) diversicolor. PeerJ 6, e5675 (2018).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
42.Kristensen, E. Ventilation and oxygen uptake by three species of Nereis (Annelida: Polychaeta). I. Effects of hypoxia. Mar. Ecol. Prog. Ser. 12, 289–297 (1983).ADS 
Article 

Google Scholar 
43.Cozzoli, F. et al. The combined influence of body size and density on cohesive sediment resuspension by bioturbators. Sci. Rep. 8, 1–12 (2018).CAS 
Article 

Google Scholar 
44.Cozzoli, F. et al. A process based model of cohesive sediment resuspension under bioturbators’ influence. Sci. Total Environ. 670, 18–30 (2019).ADS 
CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
45.Scaps, P. A review of the biology, ecology and potential use of the common ragworm Hediste diversicolor (O.F. Müller) (Annelida: Polychaeta). Hydrobiologia 470, 203–218 (2002).Article 

Google Scholar 
46.Cassidy, C., Grange, L. J., Garcia, C., Bolam, S. G. & Godbold, J. A. Species interactions and environmental context affect intraspecific behavioural trait variation and ecosystem function. Proc. R. Soc. B Biol. Sci. 287, 20192143 (2020).Article 

Google Scholar 
47.Thomsen, M. S. et al. Compensatory responses can alter the form of the biodiversity—function relation curve. Philos. Trans. R. Soc. B 286, 20190287 (2019).CAS 

Google Scholar 
48.Hale, R. et al. Mediation of macronutrients and carbon by post-disturbance shelf sea sediment communities. Biogeochemistry 135, 121–133 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
49.Karlson, K., Bonsdorff, E. & Rosenberg, R. The impact of benthic macrofauna for nutrient fluxes from Baltic Sea sediments. Ambio 36, 161–167 (2007).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
50.Thomsen, M. S. et al. Compensatory responses can alter the form of the biodiversity-function relation curve. Proc. R. Soc. B Biol. Sci. 286, 20190287 (2019).CAS 
Article 

Google Scholar 
51.Wohlgemuth, D., Solan, M. & Godbold, J. A. Species contributions to ecosystem process and function can be population dependent and modified by biotic and abiotic setting. Proc. R. Soc. B: Biol. Sci. 284, 20162805 (2017).Article 

Google Scholar 
52.Lillebø, A. I., Neto, J. M., Flindt, M. R., Marques, J. C. & Pardal, M. A. Phosphorous dynamics in a temperate intertidal estuary. Estuar. Coast. Shelf Sci. 61, 101–109 (2004).ADS 
Article 
CAS 

Google Scholar 
53.Lillebø, A. I. et al. Management of a shallow temperate estuary to control eutrophication: The effect of hydrodynamics on the system’s nutrient loading. Estuar. Coast. Shelf Sci. 65, 697–707 (2005).ADS 
Article 

Google Scholar 
54.Verdelhos, T., Cardoso, P. G., Dolbeth, M. & Pardal, M. A. Recovery trends of Scrobicularia plana populations after restoration measures, affected by extreme climate events. Mar. Environ. Res. 98, 39–48 (2014).CAS 
PubMed 
Article 

Google Scholar 
55.Hale, R., Mavrogordato, M. N., Tolhurst, T. J. & Solan, M. Characterizations of how species mediate ecosystem properties require more comprehensive functional effect descriptors. Sci. Rep. 4, 1–6 (2014).
Google Scholar 
56.Benton, T. G., Solan, M., Travis, J. M. J. & Sait, S. M. Microcosm experiments can inform global ecological problems. Trends Ecol. Evol. 22, 516–521 (2007).PubMed 
Article 
PubMed Central 

Google Scholar 
57.Bento, E. G. et al. Climate influence on juvenile European sea bass (Dicentrarchus labrax, L.) populations in an estuarine nursery: A decadal overview. Mar. Environ. Res. 122, 93–104 (2016).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
58.Martinho, F. et al. The influence of an extreme drought event in the fish community of a southern Europe temperate estuary. Estuar. Coast. Shelf Sci. 75, 537–546 (2007).ADS 
Article 

Google Scholar 
59.Solan, M. et al. In situ quantification of bioturbation using time-lapse fluorescent sediment profile imaging (f-SPI), luminophore tracers and model simulation. Mar. Ecol. Prog. Ser. 271, 1–12 (2004).ADS 
Article 

Google Scholar 
60.Schiffers, K., Teal, L. R., Travis, J. M. J. & Solan, M. An open source simulation model for soil and sediment bioturbation. PLoS ONE 6, e28028 (2011).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
61.Grasshoff, K., Kremling, K. & Ehrhardt, M. Methods of Seawater Analysis (Verlag Chemie, 1983).
Google Scholar 
62.Jones, M. N. Nitrate reduction by shaking with cadmium. Alternative to cadmium columns. Water Res. 18, 643–646 (1984).ADS 
CAS 
Article 

Google Scholar 
63.Hayward, P. J. & Ryland, J. S. Handbook of the Marine Fauna of North-West Europe. (Oxford University Press, 2017). https://doi.org/10.1093/acprof:oso/9780199549443.001.0001.64.Anderson, M. J., Gorley, R. N. & Clarke, K. R. PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods. 214 (PRIMER-E Ltd., Plymouth, UK, 2008).65.Ricotta, C. & Moretti, M. CWM and Rao’s quadratic diversity: A unified framework for functional ecology. Oecologia 167, 181–188 (2011).ADS 
PubMed 
Article 

Google Scholar 
66.Zuur, A. F. & Ieno, E. N. A protocol for conducting and presenting results of regression-type analyses. Methods Ecol. Evol. 7, 636–645 (2016).Article 

Google Scholar 
67.R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna,
Austria. (2019) https://www.R-project.org/.68.Oksanen, J. et al. vegan: Community Ecology Package. R package 2.5-6. (2019). https://CRAN.Rproject.org/package=vegan.69.Laliberté, E., Legendre, P. & Shipley, B. FD: Measuring functional diversity from multiple traits, and other tools for functional ecology. R package version 1.0-12. (2014).70.Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, 2016).MATH 
Book 

Google Scholar  More

CORRESPONDENCE
01 June 2021

French vote for river barriers defies biodiversity strategy

Simon Blanchet

 ORCID: http://orcid.org/0000-0002-3843-589X

0
&

Pablo A. Tedesco

 ORCID: http://orcid.org/0000-0001-5972-5928

1

Simon Blanchet

National Centre for Scientific Research (CNRS), Moulis, France.

View author publications

You can also search for this author in PubMed
 Google Scholar

Pablo A. Tedesco

French National Research Institute for Development (IRD), Toulouse, France.

View author publications

You can also search for this author in PubMed
 Google Scholar

Share on Twitter
Share on Twitter

Share on Facebook
Share on Facebook

Share via E-Mail
Share via E-Mail

Download PDF

Europe’s rivers are disrupted by more than one million artificial barriers, including small dams, weirs and fords (see, for example, B. Belleti et al. Nature 588, 436–441; 2020). There is strong scientific evidence that such obstructions can harm both hydrological and ecological systems, yet the French parliament has voted to leave them in place (see go.nature.com/3ck9mxq).By limiting the transfer of sediments and movement of organisms, these small barriers create a succession of reaches of warming, stagnant water that threatens freshwater biodiversity (M. R. Fuller et al. Ann. NY Acad. Sci. 1335, 31–51; 2015). Dismantling such small barriers is the most effective way to restore river connectivity and is now a worldwide objective (J. E. O’Connor et al. Science 348, 496–497; 2015).The French parliament’s decision flies in the face of the EU Biodiversity Strategy. It also has no economic justification. Most small barriers cannot generate hydroelectricity and those that can contribute less than 1% to France’s electricity (see go.nature.com/2rphjch).In our view, the fate of each barrier should be decided by balancing its ecological benefits and socioeconomic costs.

Nature 594, 26 (2021)
doi: https://doi.org/10.1038/d41586-021-01467-0

Competing Interests
The authors declare no competing interests.

Latest on:

Ecology

Trade resolution further threatens Brazil’s amphibians
Correspondence 25 MAY 21

Our radical changes to Earth’s greenery began long ago — with farms, not factories
Research Highlight 20 MAY 21

Controversial forestry experiment will be largest-ever in United States
News 20 MAY 21

Biodiversity

Trade resolution further threatens Brazil’s amphibians
Correspondence 25 MAY 21

Controversial forestry experiment will be largest-ever in United States
News 20 MAY 21

Nature-based solutions can help cool the planet — if we act now
Comment 12 MAY 21

Policy

Wanted: rules for pandemic data access that everyone can trust
Editorial 01 JUN 21

Elite US science academy expels astronomer Geoff Marcy following harassment complaints
News 27 MAY 21

Protect precious scientific collaboration from geopolitics
Editorial 26 MAY 21

Jobs from Nature Careers

All jobs

Research Fellow in Ancient Human Population Genetics
University of Tartu (UT)
Tartu, Estonia

JOB POST

Research Fellow in Ancient Metagenomics
University of Tartu (UT)
Tartu, Estonia

JOB POST

Post-doctoral Fellowship
The Royal Horticultural Society (RHS)
Woking, United Kingdom

JOB POST

Director, Division of Intramural Research (Scientific Director)
NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)
Bethesda, United States

JOB POST

Nature Briefing
An essential round-up of science news, opinion and analysis, delivered to your inbox every weekday.

Email address

Yes! Sign me up to receive the daily Nature Briefing email. I agree my information will be processed in accordance with the Nature and Springer Nature Limited Privacy Policy.

Sign up More

1.Neugschwandter, R. W. & Kaul, H. P. Sowing ratio and N fertilization affect yield and yield components of oat and pea in intercrops. Field Crops Res. 155, 159–163 (2014).Article 

Google Scholar 
2.Hu, F. et al. Low N fertilizer application and intercropping increases N concentration in pea (Pisum sativum L.) grains. Front Plant Sci. 9, 1763 (2018).PubMed 
PubMed Central 
Article 

Google Scholar 
3.Jensen, E. S., Carlsson, G. & Hauggaard-Nielsen, H. Intercropping of grain legumes and cereals improves the use of soil N resources and reduces the requirement for synthetic fertilizer N: a global-scale analysis. Agron. Sustain. Dev. 40, 5 (2020).Article 

Google Scholar 
4.Jannoura, R., Joergensen, R. G. & Bruns, C. Organic fertilizer effects on growth, crop yield, and soil microbial biomass indices in sole and intercropped peas and oats under organic farming conditions. Eur. J. Agron. 52, 259–270 (2014).Article 

Google Scholar 
5.Darch, T. et al. Inter- and intra-species intercropping of barley cultivars and legume species, as affected by soil phosphorus availability. Plant Soil 427, 125–138 (2018).CAS 
PubMed 
Article 

Google Scholar 
6.Monti, M., Pellicanò, A., Santonoceto, C., Preiti, G. & Pristeri, A. Yield components and nitrogen use in cereal-pea intercrops in Mediterranean environment. Field Crops Res. 196, 379–388 (2016).Article 

Google Scholar 
7.Scalise, A., Pappa, V. A., Gelsomino, A. & Rees, R. M. Pea cultivar and wheat residues affect carbon/nitrogen dynamics in pea-triticale intercropping: a microcosms approach. Sci. Tot. Environ. 592, 436–450 (2017).CAS 
Article 

Google Scholar 
8.Bedoussac, L. et al. Ecological principles underlying the increase of productivity achieved by cereal-grain legume intercrops in organic farming. A review. Agron. Sustain. Dev. 35, 911–935 (2015).Article 

Google Scholar 
9.Garcia, K., Doidy, J., Zimmermann, S. D., Wipf, D. & Courty, P. E. Take a trip through the plant and fungal transportome of mycorrhiza. Trends Plant Sci. 21, 937–950 (2016).CAS 
PubMed 
Article 

Google Scholar 
10.Oelbermann, M., Regehr, A. & Echarte, L. Changes in soil characteristics after six seasons of cereal–legume intercropping in the Southern Pampa. Geoderma Reg. 4, 100–107 (2015).Article 

Google Scholar 
11.Wichern, F., Eberhardt, E., Mayer, J., Joergensen, R. G. & Müller, T. Nitrogen rhizodeposition in agricultural crops: methods, estimates and future prospects. Soil Biol. Biochem. 40, 30–48 (2008).CAS 
Article 

Google Scholar 
12.Pausch, J., Tian, J., Riederer, M. & Kuzyakov, Y. Estimation of rhizodeposition at field scale: upscaling of a 14C labeling study. Plant Soil 364, 273–285 (2013).CAS 
Article 

Google Scholar 
13.Fustec, J., Lesuffleur, F., Mahieu, S. & Cliquet, J. B. Nitrogen rhizodeposition of legumes. A review. Agron. Sustain. Dev. 30, 57–66 (2010).CAS 
Article 

Google Scholar 
14.Hupe, A. et al. Get on your boots: estimating root biomass and rhizodeposition of peas under field conditions reveals the necessity of field experiments. Plant Soil 443, 449–462 (2019).CAS 
Article 

Google Scholar 
15.Parniske, M. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat. Rev. Microbiol. 6, 763–775 (2008).CAS 
PubMed 
Article 

Google Scholar 
16.Jones, D. L., Hodge, A. & Kuzyakov, Y. Plant and mycorrhizal regulation of rhizodeposition. New Phytol. 163, 459–480 (2004).CAS 
PubMed 
Article 

Google Scholar 
17.Hupe, A. et al. Even flow? Changes of carbon and nitrogen release from pea roots over time. Plant Soil 431, 143–157 (2018).CAS 
Article 

Google Scholar 
18.He, X., Xu, M., Qiu, C. Y. & Zhou, J. Use of 15N stable isotope to quantify nitrogen transfer between mycorrhizal plants. J. Plant Ecol. 2, 107–118 (2009).Article 

Google Scholar 
19.Pepe, A., Giovannetti, M. & Sbrana, C. Lifespan and functionality of mycorrhizal fungal mycelium are uncoupled from host plant lifespan. Sci. Rep. 8, 10235 (2018).ADS 
PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
20.Xiao, Y., Li, L. & Zhang, F. Effect of root contact on interspecific competition and N transfer between wheat and faba bean using direct and indirect 15N techniques. Plant Soil 262, 45–54 (2004).CAS 
Article 

Google Scholar 
21.Thilakarathna, M. S., McElroy, M. S., Chapagain, T., Papadopoulos, Y. A. & Raizada, M. N. Belowground nitrogen transfer from legumes to non-legumes under managed herbaceous cropping systems. A review. Agron. Sustain. Dev. 36, 58 (2016).Article 
CAS 

Google Scholar 
22.Meng, L. et al. Arbuscular mycorrhizal fungi and rhizobium facilitate nitrogen uptake and transfer in soybean/maize intercropping system. Front Plant Sci. 6, 339 (2015).ADS 
PubMed 
PubMed Central 

Google Scholar 
23.Shao, Z. et al. Root contact between maize and alfalfa facilitates nitrogen transfer and uptake using techniques of foliar 15N-labeling. Agronomy 10, 360 (2020).CAS 
Article 

Google Scholar 
24.Duc, G., Trouvelot, A., Gianinazzi-Pearson, V. & Gianinazzi, S. First report of non-mycorrhizal plant mutants (Myc−) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.). Plant Sci. 60, 215–222 (1989).Article 

Google Scholar 
25.Kleikamp, B. & Joergensen, R. G. Evaluation of arbuscular mycorrhiza with symbiotic and nonsymbiotic pea isolines at three sites in the Alentejo, Portugal. J. Plant Nutr. Soil Sci. 169, 661–669 (2006).CAS 
Article 

Google Scholar 
26.Jannoura, R., Kleikamp, B., Dyckmans, J. & Joergensen, R. G. Impact of pea growth and of arbuscular mycorrhizal fungi on the decomposition of 15N-labeled maize residues. Biol. Fertil. Soils 48, 547–560 (2012).Article 

Google Scholar 
27.Chalk, P. M. et al. Methodologies for estimating nitrogen transfer between legumes and companion species in agro-ecosystems: a review of 15N-enriched techniques. Soil Biol. Biochem. 73, 10–21 (2014).CAS 
Article 

Google Scholar 
28.Wahbi, S. et al. Enhanced transfer of biologically fixed N from faba bean to intercropped wheat through mycorrhizal symbiosis. Appl. Soil Ecol. 107, 91–98 (2016).Article 

Google Scholar 
29.Ingraffia, R., Amato, G., Frenda, A. S. & Giambalvo, D. Impacts of arbuscular mycorrhizal fungi on nutrient uptake, N2 fixation, N transfer, and growth in a wheat/faba bean intercropping system. PLoS ONE 14, e0213672 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
30.Fusconi, A. Regulation of root morphogenesis in arbuscular mycorrhizae, what role do fungal exudates, phosphate, sugars and hormones play in lateral root formation. Ann. Bot. 113, 19–33 (2014).CAS 
PubMed 
Article 

Google Scholar 
31.Wang, W. et al. Nutrient exchange and regulation in arbuscular mycorrhizal symbiosis. Mol. Plant 10, 1147–1158 (2017).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
32.Xue, Y. et al. Crop acquisition of phosphorus, iron and zinc from soil in cereal/legume intercropping systems: a critical review. Ann. Bot. 117, 363–377 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
33.Abdelhalim, T., Jannoura, R. & Joergensen, R. G. Mycorrhiza response and phosphorus acquisition efficiency of sorghum cultivars differing in strigolactone composition. Plant Soil 437, 55–63 (2019).CAS 
Article 

Google Scholar 
34.Louarn, G. et al. The amounts and dynamics of nitrogen transfer to grasses differ in alfalfa and white clover-based grass-legume mixtures as a result of rooting strategies and rhizodeposit quality. Plant Soil 389, 289–305 (2015).CAS 
Article 

Google Scholar 
35.Faust, S., Kaiser, K., Wiedner, K., Glaser, B. & Joergensen, R. G. Comparison of different methods to determine lignin concentration and quality in herbaceous and woody plant residues. Plant Soil 433, 7–18 (2018).CAS 
Article 

Google Scholar 
36.Baldrian, P. et al. Production of extracellular enzymes and degradation of biopolymers by saprotrophic microfungi from the upper layers of forest soil. Plant Soil 338, 1–15 (2011).Article 
CAS 

Google Scholar 
37.Wichern, F., Andreeva, D., Joergensen, R. G. & Kuzyakov, Y. Distribution of applied 14C and 15N in legumes using two different labelling methods. J. Plant Nutr. Soil Sci. 174, 732–741 (2011).CAS 
Article 

Google Scholar 
38.Turner, T. R. et al. Comparative metatranscriptomics reveals kingdom level changes in the rhizosphere microbiome of plants. ISME J. 7, 2248–2258 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
39.Yu, L., Nicolaisen, M., Larsen, J. & Ravnskov, S. Molecular characterization of root-associated fungal communities in relation to health status of Pisum sativum using barcoded pyrosequencing. Plant Soil 357, 395–405 (2012).CAS 
Article 

Google Scholar 
40.Gunina, A. & Kuzyakov, Y. Sugars in soil and sweets for microorganisms: review of origin, content, composition and fate. Soil Biol. Biochem. 90, 87–100 (2015).CAS 
Article 

Google Scholar 
41.Allison, S. D. Cheaters, diffusion and nutrients constrain decomposition by microbial enzymes in spatially structured environments. Ecol. Lett. 8, 626–635 (2005).Article 

Google Scholar 
42.Joergensen, R. G. & Wichern, F. Alive and kicking: why dormant soil microorganisms matter. Soil Biol. Biochem. 116, 419–430 (2018).CAS 
Article 

Google Scholar 
43.IUSS Working Group. WRB World reference base for soil resources 2014 (update 2015), international soil classification system for naming soils and creating legends for soil maps. World Soil Resources Reports (2015).44.Mahieu, S., Fustec, J., Jensen, E. S. & Crozat, Y. Does labelling frequency affect N rhizodeposition assessment using the cotton-wick method?. Soil Biol. Biochem. 41, 2236–2243 (2009).CAS 
Article 

Google Scholar 
45.Russell, C. A. & Fillery, I. R. P. Estimates of lupin below-ground biomass nitrogen, drymatter, and nitrogen turnover to wheat. Crop Pasture Sci. 47, 1047–1059 (1996).CAS 
Article 

Google Scholar 
46.Wichern, F., Mayer, J., Joergensen, R. & Müller, T. Evaluation of the wick method for in situ 13C and 15N labelling of annual plants using sugar-urea mixtures. Plant Soil 329, 105–115 (2010).CAS 
Article 

Google Scholar 
47.Phillips, J. M. & Hayman, D. S. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transact. Brit. Mycol. Soc. 55, 158–168 (1970).Article 

Google Scholar 
48.Brookes, P. C., Landman, A., Pruden, G. & Jenkinson, D. S. Chloroform fumigation and the release of soil nitrogen. A rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17, 837–842 (1985).CAS 
Article 

Google Scholar 
49.Vance, E. D., Brookes, P. C. & Jenkinson, D. S. An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 19, 703–707 (1987).CAS 
Article 

Google Scholar 
50.Mueller, T., Joergensen, R. G. & Meyer, B. Estimation of soil microbial biomass C in the p resence of living roots by fumigation-extraction. Soil Biol. Biochem. 24, 179–181 (1992).Article 

Google Scholar 
51.Wu, J., Joergensen, R. G., Pommerening, B., Chaussod, R. & Brookes, P. C. Measurement of soil microbial biomass C by fumigation-extraction—an automated procedure. Soil Biol. Biochem. 22, 1167–1169 (1990).CAS 
Article 

Google Scholar 
52.Hupe, A., Schulz, H., Bruns, C., Joergensen, R. G. & Wichern, F. Digging in the dirt—inadequacy of below-ground plant biomass quantification. Soil Biol. Biochem. 96, 137–144 (2016).CAS 
Article 

Google Scholar  More

1.Barrett, S. C. & Hough, J. Sexual dimorphism in flowering plants. J. Exp. Bot. 64, 67–82 (2012).PubMed 
Article 
CAS 

Google Scholar 
2.Retuerto, R., Sánchez Vilas, J. & Varga, S. Sexual dimorphism in response to stress. Environ. Exp. Bot. 146, 1–4 (2018).Article 

Google Scholar 
3.Poissant, J., Wilson, A. J. & Coltman, D. W. Sex-specific genetic variance and the evolution of sexual dimorphism: a systematic review of cross-sex genetic correlations. Evolution 64, 97–107 (2010).PubMed 
Article 

Google Scholar 
4.Bonduriansky, R. & Chenoweth, S. F. Intralocus sexual conflict. Trends Ecol. Evol. 24, 280–288 (2009).PubMed 
Article 

Google Scholar 
5.Pennell, T. M., de Haas, F. J., Morrow, E. H. & van Doorn, G. S. Contrasting effects of intralocus sexual conflict on sexually antagonistic coevolution. Proc. Natl Acad. Sci. USA 113, E978–E986 (2016).CAS 
PubMed 
Article 

Google Scholar 
6.Charlesworth, B. & Charlesworth, D. A model for the evolution of dioecy and gynodioecy. Am. Nat. 112, 975–997 (1978).Article 

Google Scholar 
7.Lloyd, D. G. & Webb, C. Secondary sex characters in plants. Bot. Rev. 43, 177–216 (1977).Article 

Google Scholar 
8.Torimaru, T. & Tomaru, N. Relationships between flowering phenology, plant size, and female reproductive output in a dioecious shrub, Ilex leucoclada (Aquifoliaceae). Botany 84, 1860–1869 (2006).
Google Scholar 
9.Delph, L. F. & Meagher, T. R. Sexual dimorphism masks life history trade-offs in the dioecious plant Silene latifolia. Ecology 76, 775–785 (1995).Article 

Google Scholar 
10.Carroll, S. B. & Delph, L. F. The effects of gender and plant architecture on allocation to flowers in dioecious Silene latifolia (Caryophyllaceae). Int. J. Plant Sci. 157, 493–500 (1996).Article 

Google Scholar 
11.Delph, L. F., Gehring, J. L., Arntz, A. M., Levri, M. & Frey, F. M. Genetic correlations with floral display lead to sexual dimorphism in the cost of reproduction. Am. Nat. 166, S31–S41 (2005).PubMed 
Article 
PubMed Central 

Google Scholar 
12.Barrett, S. C., Yakimowski, S. B., Field, D. L. & Pickup, M. Ecological genetics of sex ratios in plant populations. Philos. Trans. R. Soc. Lond. B Biol. Sci. 365, 2549–2557 (2010).PubMed 
PubMed Central 
Article 

Google Scholar 
13.Muyle, A., Shearn, R. & Marais, G. A. The evolution of sex chromosomes and dosage compensation in plants. Genome Biol. Evol. 9, 627–645 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
14.Connallon, T. & Knowles, L. L. Intergenomic conflict revealed by patterns of sex-biased gene expression. Trends Genet. 21, 495–499 (2005).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
15.Ellegren, H. & Parsch, J. The evolution of sex-biased genes and sex-biased gene expression. Nat. Rev. Genet. 8, 689–698 (2007).CAS 
PubMed 
Article 

Google Scholar 
16.Rice, W. R. Sex chromosomes and the evolution of sexual dimorphism. Evolution 38, 735–742 (1984).PubMed 
Article 

Google Scholar 
17.Charlesworth, B., Jordan, C. Y. & Charlesworth, D. The evolutionary dynamics of sexually antagonistic mutations in pseudoautosomal regions of sex chromosomes. Evolution 68, 1339–1350 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
18.Mank, J. E. The transcriptional architecture of phenotypic dimorphism. Nat. Ecol. Evol. 1, 1–7 (2017).Article 

Google Scholar 
19.Zemp, N. et al. Evolution of sex-biased gene expression in a dioecious plant. Nat. Plants 2, 16168 (2016).CAS 
PubMed 
Article 

Google Scholar 
20.Sanderson, B. J., Wang, L., Tiffin, P., Wu, Z. & Olson, M. S. Sex-biased gene expression in flowers, but not leaves, reveals secondary sexual dimorphism in Populus balsamifera. New Phytol. 221, 527–539.21.Delph, L. F. & Herlihy, C. R. Sexual, fecundity, and viability selection on flower size and number in a sexually dimorphic plant. Evolution: Int. J. Org. Evolution 66, 1154–1166 (2012).Article 

Google Scholar 
22.Golonka, A. M., Sakai, A. K. & Weller, S. G. Wind pollination, sexual dimorphism, and changes in floral traits of Schiedea (Caryophyllaceae). Am. J. Bot. 92, 1492–1502 (2005).PubMed 
Article 
PubMed Central 

Google Scholar 
23.Aloni, R., Aloni, E., Langhans, M. & Ullrich, C. I. Role of auxin in regulating Arabidopsis flower development. Planta 223, 315–328 (2006).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
24.Rocheta, M. et al. Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber. Front. Plant Sci. 5, 599 (2014).PubMed 
PubMed Central 
Article 

Google Scholar 
25.Zhao, D. & Tao, J. Recent advances on the development and regulation of flower color in ornamental plants. Front. Plant Sci. 6, 261 (2015).PubMed 
PubMed Central 

Google Scholar 
26.Moreau, C. et al. The b gene of pea encodes a defective flavonoid 3′, 5′-hydroxylase, and confers pink flower color. Plant Physiol. 159, 759–768 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
27.Hao, Z., Liu, S., Hu, L., Shi, J. & Chen, J. Transcriptome analysis and metabolic profiling reveal the key role of carotenoids in the petal coloration of Liriodendron tulipifera. Hortic. Res. 7, 1–16 (2020).Article 
CAS 

Google Scholar 
28.Hormaza, J. & Polito, V. Pistillate and staminate flower development in dioecious Pistacia vera (Anacardiaceae). Am. J. Bot. 83, 759–766 (1996).Article 

Google Scholar 
29.Boucher, L. D., Manchester, S. R. & Judd, W. S. An extinct genus of Salicaceae based on twigs with attached flowers, fruits, and foliage from the Eocene Green River Formation of Utah and Colorado, USA. Am. J. Bot. 90, 1389–1399 (2003).PubMed 
Article 
PubMed Central 

Google Scholar 
30.Manchester, S. R., Judd, W. S. & Handley, B. Foliage and fruits of early poplars (Salicaceae: Populus) from the Eocene of Utah, Colorado, and Wyoming. Int. J. Plant Sci. 167, 897–908 (2006).Article 

Google Scholar 
31.Wu, J. et al. Phylogeny of Salix subgenus Salix sl (Salicaceae): delimitation, biogeography, and reticulate evolution. BMC Evol. Biol. 15, 31 (2015).PubMed 
PubMed Central 
Article 

Google Scholar 
32.Liao, J., Cai, Z., Song, H. & Zhang, S. Poplar males and willow females exhibit superior adaptation to nocturnal warming than the opposite sex. Sci. Total Environ. 717, 137179 (2020).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
33.Dawson, T. E. & Bliss, L. Patterns of water use and the tissue water relations in the dioecious shrub, Salix arctica: the physiological basis for habitat partitioning between the sexes. Oecologia 79, 332–343 (1989).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
34.Lei, Y., Chen, K., Jiang, H., Yu, L. & Duan, B. Contrasting responses in the growth and energy utilization properties of sympatric Populus and Salix to different altitudes: implications for sexual dimorphism in Salicaceae. Physiol. Plant 159, 30–41 (2017).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
35.Ueno, N., Suyama, Y. & Seiwa, K. What makes the sex ratio female-biased in the dioecious tree Salix sachalinensis? J. Ecol. 95, 951–959 (2007).Article 

Google Scholar 
36.Jiang, H., Zhang, S., Lei, Y., Xu, G. & Zhang, D. Alternative growth and defensive strategies reveal potential and gender specific trade-offs in dioecious plants Salix paraplesia to nutrient availability. Front. Plant Sci. 7, 1064 (2016).PubMed 
PubMed Central 

Google Scholar 
37.Liao, J., Song, H., Tang, D. & Zhang, S. Sexually differential tolerance to water deficiency of Salix paraplesia-A female-biased alpine willow. Ecol. Evol. 9, 8450–8464 (2019).PubMed 
PubMed Central 
Article 

Google Scholar 
38.Saska, M. M. & Kuzovkina, Y. A. Phenological stages of willow (Salix). Ann. Appl. Biol. 156, 431–437 (2010).Article 

Google Scholar 
39.Thomas, R., Sheard, R. & Moyer, J. Comparison of conventional and automated procedures for nitrogen, phosphorus, and potassium analysis of plant material using a single digestion 1. Agron. J. 59, 240–243 (1967).CAS 
Article 

Google Scholar 
40.Arnon, D. I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1 (1949).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
41.R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, Austria, 2020).42.Wickham, H. ggplot2: elegant graphics for data analysis (springer, 2016).43.Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
44.Dai, X. et al. The willow genome and divergent evolution from poplar after the common genome duplication. Cell Res. 24, 1274–1277 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
45.Wei, S., Yang, Y. & Yin, T. The chromosome-scale assembly of the willow genome provides insight into Salicaceae genome evolution. Hortic. Res. 7, 1–12 (2020).Article 
CAS 

Google Scholar 
46.Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
47.Thimm, O. et al. MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J. 37, 914–939 (2004).CAS 
PubMed 
Article 

Google Scholar 
48.Lohse, M. et al. M ercator: a fast and simple web server for genome scale functional annotation of plant sequence data. Plant Cell Environ. 37, 1250–1258 (2014).CAS 
PubMed 
Article 

Google Scholar 
49.Oliveros, J. C. Venny. An interactive tool for comparing lists with Venn’s diagrams. 2007–2015 http://bioinfogp.cnb.csic.es/tools/venny/index.html (2016).50.Kolde, R. Pheatmap: Pretty Heatmaps. R Package Version 1.0.12. https://CRANR-project.org/package=pheatmap (2019).51.Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408 (2001).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
52.López-Ibáñez, J., Pazos, F. & Chagoyen, M. MBROLE 2.0-functional enrichment of chemical compounds. Nucleic Acids Res. 44, W201–W204 (2016).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
53.Peleg, Z. & Blumwald, E. Hormone balance and abiotic stress tolerance in crop plants. Curr. Opin. Plant Biol. 14, 290–295 (2011).CAS 
PubMed 
Article 

Google Scholar 
54.Kay, P., Groszmann, M., Ross, J., Parish, R. & Swain, S. Modifications of a conserved regulatory network involving INDEHISCENT controls multiple aspects of reproductive tissue development in Arabidopsis. N. Phytol. 197, 73–87 (2013).CAS 
Article 

Google Scholar 
55.Ditengou, F. A. et al. Characterization of auxin transporter PIN 6 plasma membrane targeting reveals a function for PIN 6 in plant bolting. N. Phytol. 217, 1610–1624 (2018).CAS 
Article 

Google Scholar 
56.Ogawa, M. et al. Gibberellin biosynthesis and response during Arabidopsis seed germination. Plant Cell 15, 1591–1604 (2003).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
57.Hedden, P. & Thomas, S. G. Gibberellin biosynthesis and its regulation. Biochem. J. 444, 11–25 (2012).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
58.Tyler, L. et al. DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis. Plant Physiol. 135, 1008–1019 (2004).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
59.Silverstone, A. L., Ciampaglio, C. N. & Sun, T. P. The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell 10, 155–169 (1998).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
60.Olszewski, N., Sun, T. P. & Gubler, F. Gibberellin signaling: biosynthesis, catabolism, and response pathways. Plant Cell 14, S61–S80 (2002).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
61.Middleton, A. M. et al. Mathematical modeling elucidates the role of transcriptional feedback in gibberellin signaling. Proc. Natl Acad. Sci. USA 109, 7571–7576 (2012).CAS 
PubMed 
Article 

Google Scholar 
62.Bévort, M. & Leffers, H. Down regulation of ribosomal protein mRNAs during neuronal differentiation of human NTERA2 cells. Differentiation 66, 81–92 (2000).PubMed 
Article 

Google Scholar 
63.Brothers, M. & Rine, J. Mutations in the PCNA DNA polymerase clamp of Saccharomyces cerevisiae reveal complexities of the cell cycle and ploidy on heterochromatin assembly. Genetics 213, 449–463 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
64.Li, C., Potuschak, T., Colón-Carmona, A., Gutiérrez, R. A. & Doerner, P. Arabidopsis TCP20 links regulation of growth and cell division control pathways. Proc. Natl Acad. Sci. USA 102, 12978–12983 (2005).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
65.Ray, S. & Pollard, J. W. KLF15 negatively regulates estrogen-induced epithelial cell proliferation by inhibition of DNA replication licensing. Proc. Natl Acad. Sci. USA 109, E1334–E1343 (2012).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
66.Halim, V., Vess, A., Scheel, D. & Rosahl, S. The role of salicylic acid and jasmonic acid in pathogen defence. Plant Biol. 8, 307–313 (2006).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
67.Zhu, F. et al. Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana. Mol. Plant. Microbe Interact. 27, 567–577 (2014).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
68.Caarls, L., Pieterse, C. M., & Van Wees, S. How salicylic acid takes transcriptional control over jasmonic acid signaling. Front. Plant Sci. 6, 170 (2015).PubMed 
PubMed Central 
Article 

Google Scholar 
69.Wang, D., Weaver, N. D., Kesarwani, M. & Dong, X. Induction of protein secretory pathway is required for systemic acquired resistance. Science 308, 1036–1040 (2005).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
70.Checker, V. G., Kushwaha, H. R., Kumari, P. & Yadav, S. Role of phytohormones in plant defense: signaling and cross talk in Molecular aspects of plant-pathogen interaction (eds Singh, A. & Singh, I.) 159–184 (Springer, 2018).71.Niki, T., Mitsuhara, I., Seo, S., Ohtsubo, N. & Ohashi, Y. Antagonistic effect of salicylic acid and jasmonic acid on the expression of pathogenesis-related (PR) protein genes in wounded mature tobacco leaves. Plant Cell Physiol. 39, 500–507 (1998).CAS 
Article 

Google Scholar 
72.Shim, J. S. et al. AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling. Plant J. 73, 483–495 (2013).CAS 
PubMed 
Article 

Google Scholar 
73.Romano, A. & Conway, T. Evolution of carbohydrate metabolic pathways. Res. Microbiol. 147, 448–455 (1996).CAS 
PubMed 
Article 

Google Scholar 
74.Akram, M. Citric acid cycle and role of its intermediates in metabolism. Cell Biochem. Biophys. 68, 475–478 (2014).CAS 
PubMed 
Article 

Google Scholar 
75.Plaxton, W. C. The organization and regulation of plant glycolysis. Annu. Rev. Plant Biol. 47, 185–214 (1996).CAS 
Article 

Google Scholar 
76.Montal, E. D. et al. PEPCK coordinates the regulation of central carbon metabolism to promote cancer cell growth. Mol. Cell 60, 571–583 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
77.Yang, J., Kalhan, S. C. & Hanson, R. W. What is the metabolic role of phosphoenolpyruvate carboxykinase? J. Biol. Chem. 284, 27025–27029 (2009).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
78.Huang, Y.-X. et al. Phosphoenolpyruvate carboxykinase (PEPCK) deficiency affects the germination, growth and fruit sugar content in tomato (Solanum lycopersicum L.). Plant Physiol. Biochem. 96, 417–425 (2015).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
79.Malone, S. et al. Phospho enol pyruvate carboxykinase in Arabidopsis: changes in gene expression, protein and activity during vegetative and reproductive development. Plant Cell Physiol. 48, 441–450 (2007).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
80.Murray, D. R. Nutritive role of seedcoats in developing legume seeds. Am. J. Bot. 74, 1122–1137 (1987).CAS 
Article 

Google Scholar 
81.Famiani, F. et al. Phosphoenolpyruvate carboxykinase and its potential role in the catabolism of organic acids in the flesh of soft fruit during ripening. J. Exp. Bot. 56, 2959–2969 (2005).CAS 
PubMed 
Article 

Google Scholar 
82.Osorio, S. et al. Alteration of the interconversion of pyruvate and malate in the plastid or cytosol of ripening tomato fruit invokes diverse consequences on sugar but similar effects on cellular organic acid, metabolism, and transitory starch accumulation. Plant Physiol. 161, 628–643 (2013).CAS 
PubMed 
Article 

Google Scholar 
83.Yuan, H., Zhang, J., Nageswaran, D. & Li, L. Carotenoid metabolism and regulation in horticultural crops. Hortic. Res. 2, 1–11 (2015).Article 
CAS 

Google Scholar 
84.Borghi, M. & Fernie, A. R. Floral metabolism of sugars and amino acids: implications for pollinators’ preferences and seed and fruit set. Plant Physiol. 175, 1510–1524 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
85.Sagawa, J. M. et al. An R2R3-MYB transcription factor regulates carotenoid pigmentation in Mimulus lewisii flowers. N. Phytol. 209, 1049–1057 (2016).CAS 
Article 

Google Scholar 
86.Tadmor, Y. et al. Genetics of flavonoid, carotenoid, and chlorophyll pigments in melon fruit rinds. J. Agric. Food Chem. 58, 10722–10728 (2010).CAS 
PubMed 
Article 

Google Scholar 
87.Chen, H. et al. A knockdown mutation of YELLOW-GREEN LEAF2 blocks chlorophyll biosynthesis in rice. Plant Cell Rep. 32, 1855–1867 (2013).CAS 
PubMed 
Article 

Google Scholar 
88.Grotewold, E. The genetics and biochemistry of floral pigments. Annu. Rev. Plant Biol. 57, 761–780 (2006).CAS 
PubMed 
Article 

Google Scholar 
89.Bennett, R. N. & Wallsgrove, R. M. Secondary metabolites in plant defence mechanisms. N. Phytol. 127, 617–633 (1994).CAS 
Article 

Google Scholar 
90.Erb, M. & Kliebenstein, D. J. Plant secondary metabolites as defenses, regulators, and primary metabolites: the blurred functional trichotomy. Plant Physiol. 184, 39–52 (2020).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
91.Falcone Ferreyra, M. L., Rius, S. & Casati, P. Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front. Plant Sci. 3, 222 (2012).CAS 
PubMed 
PubMed Central 

Google Scholar 
92.Hichri, I. et al. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J. Exp. Bot. 62, 2465–2483 (2011).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
93.Jaakola, L. & Hohtola, A. Effect of latitude on flavonoid biosynthesis in plants. Plant Cell Environ. 33, 1239–1247 (2010).CAS 
PubMed 

Google Scholar 
94.Chomicki, G. et al. The velamen protects photosynthetic orchid roots against UV‐B damage, and a large dated phylogeny implies multiple gains and losses of this function during the Cenozoic. N. Phytol. 205, 1330–1341 (2015).CAS 
Article 

Google Scholar 
95.Zhang, Y., Feng, L., Jiang, H., Zhang, Y. & Zhang, S. Different proteome profiles between male and female Populus cathayana exposed to UV-B radiation. Front. Plant Sci. 8, 320 (2017).PubMed 
PubMed Central 

Google Scholar 
96.Hideg, É., Jansen, M. A. & Strid, Å. UV-B exposure, ROS, and stress: inseparable companions or loosely linked associates? Trends Plant Sci. 18, 107–115 (2013).CAS 
PubMed 
Article 

Google Scholar 
97.Kataria, S., Jajoo, A. & Guruprasad, K. N. Impact of increasing Ultraviolet-B (UV-B) radiation on photosynthetic processes. J. Photochem. Photobiol. B: Biol. 137, 55–66 (2014).CAS 
Article 

Google Scholar  More