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Elderflora: A Modern History of Ancient Trees Jared Farmer Basic (2022)About 45 million years ago, when the Arctic was ice-free, the world’s earliest known mummified trees flourished on what is now Axel Heiberg Island in Canada’s Qikiqtaaluk Region. In 1986, palaeobotanists identified the megaflora as members of Metasequoia occidentalis, an extinct redwood species. They had been buried in silt, then frozen, their wood preserved.The lead palaeontologist “celebrated his eureka by kindling a fire with 45-million-year-old twigs and boiling water for tea time,” writes historian Jared Farmer in Elderflora, his expansive global history of grand and venerable trees. Granted, these plants had been dead since the Eocene epoch. Nevertheless, as the author describes, the incident is part of a troubling pattern in which scientists rejoice at their discovery of the ‘oldest’ tree of their time — and then destroy it.In 1957, for example, Edmund Schulman at the University of Arizona in Tucson spent the summer seeking ancient bristlecone pines in California’s White Mountains. He found three more than 4,000 years old, and named them Alpha, Beta and Gamma. Then, in the interests of tree-ring science, he chose to “sacrifice” Alpha, taking snapshots as his nephew and a colleague sawed it down. When the University of Arizona issued a press release titled ‘UA Finds Oldest Living Thing’, Farmer writes, “they say nothing about the thing being dead”.Schulman’s aim was dendroclimatology — the reconstruction of climates using tree-ring data. That lofty motive cannot be ascribed to those who, in 1881, bored a tunnel into the 2,000-year-old Wawona tree in Yosemite National Park, allowing tourists to drive their cars through the 71.3-metre-high giant sequoia (Sequoiadendron giganteum), since toppled.Arboreal legendsAs Elderflora shows, big, old trees are objects of veneration and vandalism, appearing “in the oldest surviving mythologies and the earliest extant texts”. They were associated with gods and heroes, prophets and gurus: they had pivotal roles in the Mesopotamian Epic of Gilgamesh and in the Polynesian legend of Rātā, who fells a noble tree to carve a canoe. In more recent times, European settlers “dispossessed Indigenous peoples and cleared forests with abandon”. Research shows that, for 8,000 years after the glaciers of the last ice age retreated, forests in the Midwestern United States doubled in biomass (A. M. Raiho et al. Science 376, 1491–1495; 2022). Just 150 years of industrial logging and agriculture erased this carbon accumulation.
It takes a wood to raise a tree: a memoir
“Imperial conquests and industrial revolutions relied on timber,” Farmer writes. “Wood-stock long guns for capturing lands and peoples; naval vessels with mighty masts for transporting the enslaved and the harvests of their labor.” In New Zealand, European settlers decimated the majestic kauri trees, which can live for up to 2,000 years and that once covered 1.2 million hectares of land. The trees’ 50-metre-trunks became ships’ masts; their resin was made into varnish and linoleum.Like pines, firs, spruces, cedars, cypresses and redwoods, kauri (Agathis australis) is a gymnosperm. These flowerless plants with naked seeds tend to grow slower and live longer than angiosperms, flowering plants that bear fruit. About 25 plant species — most of them conifers — can live for more than a millennium without human assistance, surviving in restricted, vulnerable habitats.Farmer also offers a global survey of ancient trees that have been protected and exalted. They include olive trees of the Levant (Olea europaea); research published this year shows that these were domesticated about 7,000 years ago for their fruit and oil (D. Langgut and Y. Garfinkel Sci. Rep. 12, 7463; 2022). In Africa, the baobab (Adansonia sp.) is both the longest-lived tree and the largest, offering shade and shelter, foods, medicines and textiles. Enslaved Africans planted baobabs in the Caribbean; some survive still. Ginkgo biloba, a species that dates back 390,000 years, survived only in China, whence it was spread around the world in the past millennium. A grove of ginkgo trees survived the atomic bombing of Hiroshima in Japan in August 1945, pushing out new buds the following spring.The planet’s current tree cover, Farmer writes, includes 3 trillion large plants covering about 30% of all land. It is, in fact, expanding. But the new cover consists mostly of shelter belts (trees planted to protect crops or animals), temperate-zone timber crops and tropical plantations of eucalyptus and palm oil. A shrinking proportion of tree cover is made up of species-rich old-growth communities.Epic loss“What would humans and nonhumans stand to lose if these survivors all died prematurely? A world of things,” Farmer writes. “Old trees sustain forest communities” with their seeds and litter. Other plants grow on them, and animals live in them. Their roots share nutrients with other organisms via underground fungi. Groups of “Old Ones” are carbon sinks. Large-scale monocultures are shorter-lived and take less greenhouse gas out of circulation.But even bygone trees of the once-tropical Arctic might offer lessons for a warming world. Palaeobotanist Hope Jahren, in her 2016 memoir Lab Girl, describes how she spent three summers on Axel Heiberg Island, digging “through a hundred vertical feet of time”. Fir, cypress, larch, redwood, spruce, pine and hemlock trees populated this lush conifer forest, with an understory of angiosperms: maple, alder, birch, hickory, chestnut, beech, ash, holly, walnut, sweetgum, sycamore, oak, willow and elm. These plants thrived even through three months of winter darkness and three of constant summer light.“Here stood one of the great forests of all time,” Farmer writes. Today, as the Arctic warms nearly four times as fast as any other place on Earth, the genomes of species related to the trees of this mummified forest might be adaptable enough for the trees to flourish in a rewarmed planet, he says. Old trees have much to teach us: we would be wise to listen. More

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The current study is the first to directly compare the differences in eukaryotic community diversity by metabarcoding eRNA and eDNA from an estuarine benthic ecosystem. This is also the first study to compare the diversity of environmental RNA and DNA using two of the most common loci examined in metabarcoding applications: COI and 18S. Only a handful of studies have used eRNA to assess diversity through metabarcoding and have demonstrated some differences in detected diversity between eRNA and eDNA in metabarcoding19,29,38. Environmental DNA has many useful applications, but one of the downfalls of DNA is that it can be detected long after an organism has inhabited an area, such as from dead organisms and even significant distances away from the source1. While the persistence of eDNA may be useful in detecting the presence of endangered, rare, or invasive species in marine systems, the persistence of eDNA, such as legacy DNA, may skew the accurate representation of community structure immediately after a disturbance event or during the exact moment of sampling21. Environmental RNA proves to be a useful tool because it is far less persistent in marine environments2,27. In the current study, RNA provides a snapshot of living organisms present in the mesocosms at the time of sampling, compared to DNA which detects past and present organisms in the sample.Benthic ecosystems often have high biodiversity because they are dynamic environments with various substrates favorable for hosting communities39. To date, there is no one loci and associated primer pair that will effectively detect all eukaryotic organisms, and in particular metazoans, so it is beneficial to use multiple loci when looking for a variety of taxa6. Previous marine eDNA studies have demonstrated the preferred method of using two loci to achieve comprehensive metabarcoding for taxonomically diverse environments5,30,40. The current study further demonstrates the need to use at least two different loci for targeted PCR and sequencing to truly capture the wide diversity in rich systems, such as the top oxygenated layer of the marine benthos. Our results demonstrate the types of organisms detected using the 18S loci vary widely from the organisms detected by COI. The 18S loci also detected a higher number of organisms than using the COI loci. The 18S allowed greater detection of metazoans whereas COI was useful in the detection of Oomycota (i.e., a eukaryotic microorganism that resembles fungi) protozoa. Protozoa are often food sources for meiofauna41; therefore, using COI for protozoan detection and 18S for metazoan detection showcases multiple trophic levels present in the mesocosms of the current study. Utilizing multiple loci not only broadens taxonomic diversity detection but can also highlight multiple trophic levels for understanding food webs originating in benthic environments15.A study evaluating arctic benthic diversity similarly found that COI detected fewer taxa than 18S using eDNA metabarcoding, and there was only ~ 40% taxa overlap between markers at the class level42. In the current study, the COI marker using both nucleic acid templates yielded a higher percentage of unassigned taxa after filtering for presence in the majority of mesocosms compared to 18S. A possible explanation for the low metazoan detection by COI may be that the unassigned taxa are metazoans rather than more SAR organisms. Additionally, singly detected metazoans were filtered out of the analysis if they were not detected in at least 4 mesocosms. For example, one type of amphipod was detected in only 3 mesocosm by RNA using the COI marker, but was not detected in any mesocosms using DNA. Therefore, the amphipod observation was excluded from the COI results in Fig. 3 and Table S3 because it was not found in at least 4 mesocosms.Overall, RNA provides a broader assessment of benthic community structure than DNA, particularly when using two loci/ markers for sequencing. In the current study, we used nuclear 18S ribosomal RNA and DNA and mitochondrial COI RNA and DNA sequences as markers for metabarcoding. The number of copies of ribosomal RNA per cell is higher than the copies of ribosomal DNA, and the ratio of RNA: DNA is higher in single-cell organisms, such as protists43. The higher number of unique ASVs detected using eRNA is likely attributed to the higher number of RNA copies of each marker in small, single-cell organisms successfully amplified during PCR, therefore making rare organisms easier to detect. The DNA of highly abundant or higher biomass organisms may “drown out” (i.e., mask) the sequences from lower abundance and biomass organisms during PCR amplification, thus resulting in lower detected α-diversity. The increased detection of ciliates and protozoa using eRNA are consistent with other recent eRNA metabarcoding results that found higher ciliate and protozoan diversity compared to eDNA using the same Uni18S primers19. Positive correlations between organism biomass and sequence copy numbers have been demonstrated for DNA metabarcoding conducted on invertebrate species44. In the current study, RNA allowed for the detected of both larger meiofauna and smaller microfauna, which is optimal for assessing true biodiversity with molecular assays. Chaetonotida, a type of gastrotrich, was the only taxa detected in 4 of the mesocosms using DNA that was not found with RNA. It is possible the chaetonotida may have died during the experiment due to sensitivities to new environmental conditions, and therefore were not detected with RNA. The temperature of the flowing seawater in the mesocosms was approximately 18 °C, and many marine chaetonotida prefer 23–28 °C and high organic matter substrate45.Previous studies have compared the accuracy of conventional morphological identification to molecular metabarcoding methods for assessing biodiversity. In estuarine-specific studies, metabarcoding methods are able to detect the majority of taxa identified with traditional methods and often detected higher species richness, or higher numbers of unique organisms, that was not found conventionally10,46,47. The limiting factor for higher resolution of taxonomic identification is the availability of species-specific sequences in barcoding databases48. However, barcoding databases are becoming more robust as an increasing number of researchers contribute high quality sequencing data to databases7. Therefore, eRNA metabarcoding techniques perform similarly to conventional morphological methods, and may even uncover higher biodiversity in systems like estuaries where meiofauna have been historically understudied and identified.Although eRNA α-diversity is higher compared to eDNA, there is some overlap between ASVs detected with eRNA and eDNA. The higher percentage of overlap between eDNA and eRNA ASVs is predominantly seen in the ASVs detected in all 7 of the mesocosms. The increased overlap in ASVs detected in all 7 mesocosms compared to the relatively lower overlap of ASVs found in only 1 of the 7 mesocosms is due to the filtering of random organisms found in only 1 mesocosm. Detection of a unique organism in a single mesocosm is likely not representative of the sample community and filters potential artifacts that may be introduced during cDNA synthesis from eRNA. However, all uniquely identified ASVs are used in the β-diversity analysis, so the higher number of unique ASVs detected from eRNA are likely driving the significant differences observed between eRNA and eDNA β-diversity. It is possible that using eRNA could increase the statistical power of a study design compared to eDNA because eRNA detects a higher number of ASVs. It is unlikely that a higher number of RNA ASVs could be due to splice variants contributing to unique sequences because the amplified regions of both markers do not contain introns. Therefore, no splicing of transcripts would be expected. Thus, the detected DNA ASVs are from living organisms in the mesocosms and the higher diversity of ASVs detected from RNA demonstrate that RNA is a more suitable option for assessing diversity of living organisms.It is evident that the mesocosms in the current study were rich with meio- and microfauna due to the number of unique organisms and broad diversity of different taxa. It is likely that collecting samples for nucleic acid extraction directly from the field site may result in higher diversity because there is no mechanical disturbance during the laboratory acclimation period and the presence of other organisms in the system, such as fish, macroinvertebrates, or birds. eDNA molecular abundance in samples has been shown to correlate to actual organismal abundance in laboratory environments, but the same correlation is not as apparent in field samples, which is likely due to a variety of collection and processing methods49. eRNA may be the better nucleic acid template for field collection once flash frozen, especially for the detection of protozoans. Future studies will compare the diversity of eukaryotic communities detected using eDNA and eRNA collected directly from the field rather than from sediment core mesocosms. Repeated sampling from a field site may help reduce transient eRNA detection when establishing accurate baseline community composition in field-based biomonitoring studies.Recently, meiofaunal organisms and communities are being explored as bioindicators, which are organisms whose presence are indicators for environmental stress and pollution17. For example, lower meiofaunal diversity and abundance is associated with higher pollution in harbors, and the presence of some genera of nematodes are correlated with higher concentrations of polycyclic aromatic hydrocarbons because they are more tolerant to pollution50. A previous study that used field-collected mesocosms from the same location as our current study found similar phyla detected with a metabarcoding approach; the majority of the sequences detected in benthic communities were from nematodes, arthropods, and the microfaunal SAR clade10. Similarly, the majority of the ASVs detected from the current study also corresponded with nematodes, arthropods, and the SAR clade, as well as other commonly detected meiobenthic organisms, such as polychaetes and Homalorhagida (i.e., mud dragons). A recent study exposed a benthic foraminiferal community to chromium, and found that eRNA metabarcoding was more robust for detecting changes in diversity at lower chromium concentrations compared to eDNA51. The eRNA metabarcoding method used in the current study detected meiofaunal taxa typical of marine or estuarine environments. Therefore, eRNA metabarcoding may be useful for efficiently identifying bioindicator species or taxa impacted by exposures to different contaminants and environmental stressors to aid with management of aquatic systems. Another advantage of eRNA is that RNA provides functional information about how organisms response to stress through altered transcription of activated pathways. eRNA will likely be a more powerful tool than eDNA because it allows for the detection of both bioindicator species and, in the future with increase development of genetic databases, environmental detection of biomarkers of stress through increased transcription of response genes.Many academic researchers are adopting molecular methods using High Throughput Sequencing as the future of biomonitoring surveys; however, few regulatory and environmental management organizations/ agencies have adopted metabarcoding into their routine biomonitoring practices for regulatory purposes. In marine benthic communities, metabarcoding provides a comprehensive assessment of diversity and is useful for detecting a broader array of organisms in biomonitoring surveys especially when using two markers47. eDNA is also useful for monitoring discrete communities (i.e., benthic versus pelagic)52, so it is possible that using eRNA could provide vital information about living organisms in specific environments compared to eDNA. Metabarcoding sequencing and bioinformatic approaches for benthic environments vary among studies, thus requiring some standardization between methods to further advance the use of metabarcoding in conservation and regulatory applications30,53.Like eDNA, some advancements must be made with eRNA to be used as a quantitative tool in molecular ecology. Validation of eDNA metabarcoding for assessing relative abundance of species is rapidly progressing by correlating laboratory studies of DNA shedding with field experiments1,54. Similar validation techniques can be used to develop eRNA metabarcoding as a quantitative or semi-quantitative method. There is growing interest in optimizing eRNA extraction protocols from different types of environmental media to standardize the use of eRNA in downstream molecular applications55. Thus, standardizing eRNA protocols will help with integration into environmental management toolkits for regulatory purposes.RNA poses unique barcoding challenges compared to DNA because the number of RNA transcripts from a gene are not always present in the same proportion compared to the gene copy number per genome (i.e., one DNA copy per cell), especially for differentially expressed genes. However, one possible way to work around this issue is to utilize constitutively expressed marker loci where transcription is generally stable and unaffected by environmental stressors, such as those used as reference genes for quantitative real-time PCR49. Fortunately, many loci chosen for metabarcoding purposes fit this criterium; the transcription of 18S and COI remain steady within the cell regardless of environmental stress.Collecting sediment cores from the environment and bringing them into controlled laboratory settings for community analysis through eRNA metabarcoding is a powerful tool that opens opportunities for this method to be used in a broad range of fields. For instance, field-collected mesocosms could be used in controlled settings to investigate the effects of individual or mixtures of toxicants on entire community and population-level outcomes. Marine sediments are often the ultimate sink for environmental contaminants, such as organic pollutants , heavy metals56, and plastic particles57, yet few studies investigate actual community-level changes in contaminant exposures. Marine benthic environments have high biodiversity, but the breadth of diversity in micro- and meiofaunal organisms is often understudied because traditional morphological methods are immensely time consuming. Sediment core mesocosms could also be used to understand the effects of global climate change stressors, such as fluctuating temperatures, surface water salinity and pH, on communities as well as conventional and emerging contaminants in combination with climate change stressors. Additionally, this method could also be used to understand how communities respond to a significant disturbance event or smaller series of stressors, which are often difficult to measure in environmental settings32,58. In these applications, eRNA is favorable for constructing community composition at a specific moment of the experiment to better regulate anthropogenic causes of environmental stress. More

Our findings indicated that mine water had a certain inhibitory effect on ryegrass seed germination, and the intensity of this inhibitory effect increased with increased mine water proportion. These effects were mainly reflected as changes in germination potential. Concretely, irrigation with mine water prolonged the germination of ryegrass seeds but had no significant effect on germination rate. Min Zhu et al. found that recycled water inhibited the seed germination of turfgrass, and this effect became more notorious when the concentration of reclaimed water increased. This was likely because the water contained salt ions, heavy metal ions, and E. coli, all of which are known to affect seed germination22. The mine water was taken from the Laohutai mining area, where the water composition and quality are good. Therefore, mine water did not significantly affect seed germination and the seeds maybe germinate normally if given sufficient time.The physiological and photosynthetic characteristics of ryegrass were impacted by the mine water, and the intensity of inhibition increased with higher mine water proportions. When the ratio of mine water to clean water reached a certain proportion (1:2, A1 and B1), the physiological and growth characteristics of ryegrass were improved to a certain extent. When only mine water was used for irrigation, the indices were significantly suppressed. In contrast, mixing clear water with mine water for irrigation promoted the physiological characteristics of the plants, as well as photosynthesis. This was likely because the mineral content of mine water is higher. However, mine water not only contains elements needed for plant growth but also some elements and ions that have inhibitory effects on plant growth. Therefore, the quality of ryegrass growth were suppressed when irrigating only with mine water. In contrast, after mixing the mine water with clean water, the concentrations of certain substances that produce adverse effects are diluted, and the mixed irrigation water promoted ryegrass growth in appropriate proportion.A certain concentration of heavy metal elements will affect the absorption of essential elements by plants and produce antagonism, and high concentration can directly lead to plant death. Heavy metal stress affects chlorophyll content through two aspects: Heavy metal destroys enzymes needed for chlorophyll synthesis, affects plant chlorophyll synthesis, and then inhibits plant photosynthesis23. The second is the destruction of chloroplast structure and cell membrane24,25,26. In the treatment of high concentrations of heavy metals, the chlorophyll content of plants is significantly reduced due to the inhibition of chlorophylase or aminolevulinic acid dehydrase, thus inhibiting plant photosynthesis27. The heavy metal threat forcing stimulates the formation of reactive oxygen species that convert fatty acids into toxic lipid peroxides, which damage to plant cells28,29,30. Heavy metal stress can induce a lot of activity in plants sexual oxygen and inhibit the normal metabolism of plants, causing membrane lipid peroxidation and increased plasma membrane permeability31, 32. Low concentration of heavy metal stress will stimulate the protective mechanism of plants, so low concentration of stimulation will not damage plants, on the contrary, may help plant growth. Heavy metal stress causes water loss in plants, and a certain amount of proline can be produced to regulate the water balance of plant cells and reduce the damage degree of plant cells33. SOD, POD and CATT are important antioxidant enzymes in plants, which can scavenge excessive free radicals. The synergistic action of three enzymes can protect plants from free radical damage. When the concentration of heavy metals was low, the activity of protective enzymes increased under the induction of reactive oxygen radicals. However, with the increase of stress degree, the activities of SOD, POD and CAT decreased, which eventually led to the persecution of plant cells34. These conclusions are consistent with the results of this paper. When mine water was mixed with clear water at a ratio of 1:2, heavy metal stress stimulated the protective mechanism of ryegrass most appropriately, and improved plant quality and resistance. On the contrary, when the proportion of mine water increased, the physiological characteristics and quality of ryegrass plants were inhibited to different degrees.Precious Nneka Amori et al. studied physiological traits of leaves and Silverbeet using treated wastewater, the results show that the biomass of plants watered with only the treated wastewater were more than 50% higher than the yield in tap water control and plants exhibited high degree of root foraging1. Libutti et al. irrigated tomato and broccoli with purified agro-industrial effluent and reported that yield and quality traits of agricultural products were not affected35. Radish was grown using a reclaimed synthetic textile wastewater treated in an anoxic-aerobic photobioreactor, and the dry weight, leaf number and leaf area of plant harvest were 49, 19.2 and 62% higher than the growth performance in freshwater irrigation36. FU et al. studied four native Chenopodiaceae plants of Halogeton glomeratus, Kochia scoparia, Suaeda glauca and Chenopodium glaucum in Jinchang area northwest China, from their changes of net photosynthetic rate (Pn), Stomatal conductance (Gs), transpiration rate (Tr). chlorophyll content (Chl), malondialdehyde (MDA), soluble protein (SP), proline (Pro) and antioxidant enzymes activity under the treatment of farmland soil (T1) and sedendary soil mixed with tailing (1:1, T2), they concluded that under T2 treatment, Pn, Gs, and Tr of Halogeton glomeratus and Kochia scoparia were decreased , the other six indexes were increased significantly. Gs, Tr, MDA, Pro, and SOD increased, yet CAT, Chl and Pn of Suaeda glauca decreased significantly, respectively. Pn, Gs, and Tr of Chenopodium glaucum decreased significantly, while SP, POD increased significantly37. Our results also indicated that mine water irrigation had significant effects on soil characteristics. At higher mine water ratios, the soil conductivity increased exponentially, the pH decreased gradually, the content of K+, Na+, Ca2+ and Mg2+ increased, and the content of N, P and K also increased gradually. In contrast, the clean water and mine water mixture had little effect on the soil properties. This was because the salt and metal ions in mine water migrate to the soil during the irrigation process, which significantly changes the soil properties. As a result, the concentration of salt in the soil increased and soil acidity also increased. After mixing with clean water, the concentration of salt decreases, and the influence on the soil matrix weakened. These results also indicated that the growth, physiological, and photosynthetic effects of ryegrass in the pot experiments were better than those in soilless culture, because there were many other organic materials and inorganic ions in soil that could promote growth, whereas the plants in the hydroponic system lacked other nutrients that benefit plant growth. Many existing studies have shown that mine drainage or other wastewater can improve the quality and yield of one or more kinds of plants to different degrees after certain treatment, but some studies also show that the reclaimed water used for irrigation will cause harm to plants, soil and even human health.Jinfang Yang et al. reported that long-term irrigation with mine water significantly reduced the soil respiration rate and soil enzyme activity. Mine water irrigation also significantly inhibited wheat plant height, leaf area, chlorophyll content, and photosynthetic rate, and wheat production was also markedly reduced38. Jianjun Cha found that acidic mining waste water can reduce the pH of the soil profile and increase its electrical conductivity39. Junhao Qin et al. found that if treated mine water is used as an irrigation water source, acidic substances may still be introduced into the soil. This inhibits plant growth and may also enhance leaching of some trace elements in the soil to shallow aquifers, resulting in groundwater pollution40, 41. The results of this study are consistent with the above conclusions, that is, directly irrigating with mine water can significantly inhibit plant growth and photosynthesis, thus affecting the quality of ryegrass plants. MA et al. studied the effects of irrigation with mine wastewater on the physiological characters and heavy metals accumulation of winter wheat. It shows that irrigation with mine wastewater had negative effects on the winter wheat growth and grain yield. At anthesis stage, the leaf area, dry mass per stem, root activity and net photosynthetic rate of winter wheat in treatments were significantly lower and the plant height and leaf chlorophyll content was decreased. In addition, the heavy metals (Cr, Pb, Cu and Zn) contents in the grain of winter wheat under mine wastewater irrigation were significantly higher than those in control, it suggested that the irrigation with mine water could result in the heavy metals accumulation in wheat grain42. A large number of studies have shown that direct use of mine water for irrigation will have a negative impact on soil and plants, but this study found that after a certain processing of mine water (mine water was mixed with clear water in a ratio of 1:2) used for irrigation does not significantly alter soil properties, but can increase plant yield and quality, it will be meaningful to mine water reuse, soil utilization around the mining area and the agriculture.The conclusions of this study are based on mine water from Fushun mining area in Northeast China, but the effects of mine water on plants from other mining areas are uncertain. At the same time, ryegrass, a cold-season turfgrass, is only selected in this study. If it is other kinds of plants, how they respond to mine water irrigation needs further study. What are the effects of mine water irrigation on plants other than ryegrass that need further study. Moreover, this study was only a short-term experiment, and the effects of mine water on the properties of the soil matrix cannot be generalized. Indoor experiments can be regularly watered to maintain moisture, indoor temperature is relatively fixed, while the natural environment is a lot of uncertainty and uncontrollable. Would the results of a small-scale pot experiment in a controlled environment be different if it were applied to a field where there are many uncertainties about soil properties and atmospheric conditions? Long-term field experiments must also be conducted to confirm our findings in more realistic conditions. The use of mine water resources not only has environmental and social benefits but could also bring economic benefits43. This study demonstrated that mine water can be used in ecological restoration and agricultural irrigation in mining areas, and is therefore of great significance to environmental restoration. More

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