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    Orbit-to-ground framework to decode and predict biosignature patterns in terrestrial analogues

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    No impact of nitrogen fertilization on carbon sequestration in a temperate Pinus densiflora forest

    SettingThis study was conducted in approximately 40-year-old naturally regenerated P. densiflora stands in Wola National Experimental Forest in Gyeongnam province in South Korea (35°12′ N, 128°10′ E; Table 1). The productivity of this forest is low, with a dominant tree height of 10 m at 20 years of age. Over the last 10 years, the mean annual precipitation was 1490 mm, of which one third fell during summer (July–August), and the mean temperature was 13.1 °C. The vegetation growing season generally lasts for approximately 200 days, extending from early April to October. The soil texture is a silt loam originating from sandstone and shale (clay 13.0 ± 0.8%, silt 44.1 ± 1.3%, sand 42.9 ± 1.6%; n = 18). The given texture results in volumetric water contents at 13.4 ± 0.7% (m3 m−3) at permanent wilting point (1500 kPa) and 40.7 ± 1.2% at field capacity (10 kPa)55. The understory is covered with Lespedeza spp., Quercus variabilis, Q. serrata, Smilax china, and Lindera glauca.In 2010, we selected two adjacent P. densiflora stands approximately 100 m apart from each other (180 m and 195 m above sea level, on slopes of 15° and 33°, both stands face south). Following a completely randomized design, we established nine plots (10 × 10 m2 with a 5 m untreated buffer) within each stand, of which three were randomly assigned to annual NPK fertilization, three to PK fertilization, and the rest to a control treatment without fertilization. The fertilizer, composed of urea, fused superphosphate and potassium chloride (N3P4K1) or P4K1 was added manually by deposition on the forest floor for 3 years in April 2011, April 2012, and March 2013. Over these 3 years, the NPK plots received 33.9 g N, 45 g P, and 11.1 g K m−2, while the PK plots received 45 g P and 11.1 g K m−2.Tree and stand measurementsThe standing biomass of trees was estimated using a combination of site-specific allometric equations based on destructive harvesting56 and repeated measurements of the dimensions of all trees in each plot (5–18 trees plot−1). The stem diameter at 1.2 m (D) was measured for all trees in each plot for which D was ≥ 6 cm. Selecting a representative tree in size for each plot within the 4 × 4 m2 center of the plot, we measured the tree height (H) and crown base for the representative trees. Measurements were performed in April and September 2011, September 2012–2014, and October 2021. We observed no effect of fertilization on the relationship between D and H or between D and crown base, so we assumed no effect on the allometric functions for foliage or branch biomass. A dendrometer band (Series 5 Manual Band, Forestry Suppliers Inc., Jackson, MS, USA) was installed on 18 representative trees (one per plot) to monitor radial growth monthly.Three 0.25 m2 circular litter traps were installed 60 cm above the forest floor in each plot in April 2011. Litter was collected at 3-month intervals between June 2011 and March 2015. The litter from each trap was transported to the laboratory and then oven-dried at 65 °C for 48 h. All dried samples were separated into needles, bark, cones, branches, and miscellaneous components, and weighed separately.In September 2014, we estimated the biomass of understory vegetation, separately for woody plants and herbaceous plants. All woody plants  More

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    Ontogenetic changes in the body structure of the Arctic fish Leptoclinus maculatus

    Meyer Ottesen, C. A. et al. Early life history of the daubed shanny (Teleostei: Leptoclinus maculatus) in Svalbard waters. Mar. Biodivers. 41(3), 383–394 (2011).Article 

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    Extinction drives a discontinuous temporal pattern of species–area relationships in a microbial microcosm system

    Preparation of the pao cai soupFirst, 35 kg of white radish (Raphanus sativus), 35 kg of cabbage (Brassica oleracea), 2 kg of chili pepper (Capsicum frutescens), 1 kg of ginger (Zingiber officinale), 1 kg of peppercorns (Zanthoxylum bungeanum), 2.5 kg of rock sugar, and 210 kg of cold boiled water (containing 6% salt) were divided into six ceramic jars. After 7 days of natural fermentation at room temperature, the pao cai was filtered out with sterile gauze to obtain 200 kg of pao cai soup. To ensure an even distribution of microorganisms in the soup, the soup was mixed well and then left to rest for 12 h, the supernatant was taken, and the soup was left to rest for 12 h again.The plants used in this study were cultivated vegetables which purchased from the vegetable market at the study site. All local, national or international guidelines and legislation were adhered to in the production of this study.Establishment of the microcosm systemSeventy-eight for each size of 10 ml, 20 ml, 50 ml, 100 ml, 250 ml, 500 ml, and 1000 ml sterile glass culture flasks were filled with pao cai soup, the bottle mouth was sealed with sterile sealing film, and the bottle was capped without leaving any air (Fig. 1). Each flask became a microcosm and was cultured in a 25 °C incubator.Figure 1Schematic diagram of the establishment of the microcosmic system.Full size imageSample collectionBefore the microcosm system was established, a sample of well-mixed pao cai soup was taken as a reference to establish background biodiversity. The microbial community dynamics should change the fastest at the beginning of the microcosm system establishment and gradually become slower over time. Considering the workload and cost, this study collected samples daily for 1–10 day after the establishment of the microcosm and then collected every 2 days for 10–30 day and every 5 days for 30–60 day. Three different microcosms of the same volume were established. Monitoring was carried out for 60 days, and a total of 546 samples of 7 volumetric gradients were obtained at 26 time points. At the time of sampling, the pao cai soup in the microcosm was mixed, and 50 mL of sample (10 mL of sample was collected for microcosm systems with a volume of less than 50 mL) was collected. The sample was centrifuged at 8000 rpm for 10 min, the supernatant was collected for pH determination, and the pellet was stored in a − 80 °C freezer.Microbial analysesMicrobial DNA was extracted from pao cai samples using the E.Z.N.A.® Soil DNA Kit (Omega Biotek, Norcross, GA, U.S.) according to the manufacturer’s protocols. For bacteria, we targeted the V3-V4 region of the 16S ribosomal RNA (rRNA) gene, using the 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primer pairs. For fungi, we targeted the ITS1-1F region of the nuclear ribosomal internal transcribed spacer region (ITS rDNA) gene, using ITS1-1F-F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS-1F-R (5′-GCTGCGTTCTTCATCGATGC-3′). PCRs were performed in triplicate in a 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The PCR program for the 16S rRNA gene was as follows: 3 min of denaturation at 95 °C; 27 cycles of 30 s at 95 °C, 30 s of annealing at 55 °C, and 45 s of elongation at 72 °C; and a final extension at 72 °C for 10 min. For the ITS1-1F region, the PCR program was as follows: samples were initially denatured at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, primer annealing at 50 °C for 30 s, and extension at 72 °C for 30 s. A final extension step of 5 min at 72 °C was added to ensure complete amplification of the target region. The resulting PCR products were extracted from a 2% agarose gel, further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, Madison, WI, USA).Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 300) on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) according to standard protocols. The analysis was conducted by following the “Atacama soil microbiome tutorial” of QIIME2 docs along with customized program scripts (https://docs.qiime2.org/2019.1/). Briefly, raw data FASTQ files were imported in the QIIME2 system using the qiime tools import program. Demultiplexed sequences from each sample were quality filtered, trimmed, denoised, and merged, and then the chimeric sequences were identified and removed using the QIIME2 DADA2 plugin to obtain the feature table of amplicon sequence variants (ASVs)24. Compared with traditional OTU that clusters at 97% similarity, ASV has higher accuracy, equivalent to 99% similarity clustering. The QIIME2 feature-classifier plugin was then used to align ASV sequences to the pretrained GREENGENES 13_8 99% database (trimmed to the V3-V4 region bound by the 338F/806R primer pair for bacteria) and UNITE database (for fungi) to generate the taxonomy table25. Any contaminating mitochondrial and chloroplast sequences were filtered using the QIIME2 feature-table plugin. Based on the sequence number of the lowest sample, perform the resampling to make the sequence number equal for each sample. Due to the random nature of sequencing, ASVs specific to each sample in this study were present. To reduce the uncertainty introduced by the sequencing process, we filtered out rare ASVs with less than 0.001% of the total sequence volume.Data analysisIn this study, the data of fungi and bacteria were integrated and analyzed, and all microbial diversity appearing in the text represent the sum of all fungi and bacteria. Species richness is equal to the number of taxa, which is equal to the total number of all bacterial and fungal ASVs. The vegan package in R 4.2.1 was used to calculate the species richness of each sample based on the ASV feature table26. Using flask volume instead of area, SAR fitting was performed using a semi-logarithmic model, and its significance was tested. The semi-logarithmic model is the function S = c + b*logA, where S is species richness, A is area (in this case, volume is used instead), and b and c are fit parameters27.The microcosmic system in this study is hermetically sealed, and all microorganisms originate from a single portion of well-mixed paocai soup (ie species pool). The speciation process in the 60-day experimental system should be negligible due to the short experimental period. The extinction rate of a microcosm system is equal to the number of ASVs lost in the microcosm system compared to the species pool divided by the total number of ASVs in the species pool. The extinction rate is the number of extinct ASVs in each system compared to the species pool. Pearson correlation analysis was performed with volume as the independent variable and extinction rate as the dependent variable to determine the correlation between volume and extinction rate at each time point. When microorganisms of a microcosmic system disappear entirely or cannot be detected, the microcosm is recorded as an annihilated microcosm. The annihilation rate at a time point is equal to the number of microcosms annihilated at that time, divided by the total number of microcosms. The difference between the extinction rate and annihilation rate defined in this paper is that the extinction rate is for ASVs within each sample, and the annihilation rate is for microcosmic system at each sampling time point. The two indicators jointly characterize the local extinction of microorganisms from different perspectives. Non-linear regression with a bell-shaped form was performed with time as an independent variable and pH and annihilation rate as dependent variables, and regression lines were plotted based on R 4.2.1.According to the taxonomy table, bacterial ASVs were divided into acid-producing and non-acid-producing categories, and their extinction rates were calculated separately. The agricola, ggplot2, vegan and ggpubr packages were used to draw alpha diversity box plots and perform the Wilcoxon rank sum test for differences between groups26,28,29,30. Non-metric multidimensional scaling (NMDS) analysis was performed with the vegan package based on Bray–Curtis dissimilarity. In addition, the potential Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologue (KO) functional profiles of microbial communities were predicted with PICRUSt31. Resistance-related genes were screened using the gene function predictions. The relationship between the relative abundance of resistance-related genes and the volume of the microcosm was analysed by Pearson correlation, and a forest map was plotted to present the results. More

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    Astrobiologists train an AI to find life on Mars

    Artificial intelligence (AI) and machine learning could revolutionize the search for life on other planets. But before these tools can tackle distant locales such as Mars, they need to be tested here on Earth.A team of researchers have successfully trained an AI to map biosignatures — any feature which provides evidence of past or present life — in a three-square-kilometre area of Chile’s Atacama Desert. The AI substantially reduced the area the team needed to search and boosted the likelihood of finding living organisms in one of the driest places on the planet. The results were reported on 6 March in Nature Astronomy1.Kimberley Warren-Rhodes, a senior research scientist at the SETI Institute in Mountain View, California, and lead author on the paper, has been chasing biosignatures since the early 2000s, when she realized how few tools existed to study the biology of other planets. She wanted to combine her background in statistical ecology with emerging technologies such as AI to help mission scientists, “who are under a lot of pressure to find biosignatures” but tightly constrained in how they do so. Rovers that are controlled remotely from Earth, for example, can travel only limited distances and collect relatively few specimens, placing a premium on sampling locations that are the most likely to yield life. Mission scientists base these predictions in part on Mars analogues on Earth, where scientists scour extreme habitats to determine how and where living organisms thrive.Searching for lifeBeginning in 2016, Warren-Rhodes’ group travelled to the high, parched plateau of the Atacama Desert — a proposed Mars analogue at an elevation of around 3,500 metres in the Chilean Andes — to search for rock-dwelling, photosynthetic organisms called endoliths. To fully characterize the environment, the researchers collected everything from drone footage to geochemical analyses to DNA sequences. Together, this data set mimics the types of information researchers are collecting on Mars with orbital satellites, drones and rovers.Warren-Rhodes’ team fed its data into an AI-based convolutional neural network (CNN) and a machine-learning algorithm that in turn predicted where life was most likely to be found in the Atacama.

    Aerial view (left) and ground view from a rover of a biosignature probability map of the same area.Credit: M. Phillips, K. A. Warren-Rhodes & F. Kalaitzis

    By targeting their sample collection on the basis of AI feedback, the researchers were able to reduce their search area by up to 97% and increase their likelihood of finding life by up to 88%. “At the end, you could plop us down, and instead of wandering around for a long time, it would take us a minute to find life,” Warren-Rhodes says. Specifically, the team found that endoliths in the Atacama were most often found in a mineral called alabaster — which is porous and retains water — and tended to aggregate in transitional areas between various microhabitats, such as where sand and alabaster crystals abut one another.“I’m very impressed and very happy to see this suite of work,” says Kennda Lynch, an astrobiologist at the Lunar and Planetary Institute in Houston, Texas, who studies biosignatures. “It’s really cool that they can show some success with an AI to help predict where to go and look.”Graham Lau, an astrobiologist at the Blue Marble Space Institute of Science who is based in Boulder, Colorado, worked on another Mars analogue in the Canadian Arctic as a graduate student, to study how biology influences the formation of rare minerals that can serve as biosignatures on other planets. “Ever since I first read Frank Herbert’s Dune as a young child, I was struck by this idea of applying ecology to planets,” he says. But up until the last decade or so, the tools and data weren’t available to address such questions with scientific rigour. “The place where we have almost unlimited data possibilities is through these orbital observations and drone imaging,” he says, “and I do see this paper as being one of many pieces along the pathway to doing these larger analyses.”Deceptively simpleThe new method will need to be verified across multiple ecosystems, Lau and Lynch say, including those with more complex geology and greater biodiversity. The Atacama, Lau notes, is relatively simple in terms of the habitats and the types of life that are likely to be found there. And on Mars, the high level of ultraviolet radiation striking the planet’s surface means that scientists might need to detect clues that hint at life below ground.

    NASA’s Perseverance rover collected its first rock sample from an area in Mars’ Jezero Crater.Credit: NASA/JPL-Caltech/ASU/MSSS

    Ultimately, Warren-Rhodes says she would like to see a comprehensive database of different Mars analogues that could feed valuable information to mission scientists planning their next sampling run. Her team’s advance, she adds, might appear “deceptively simple” to anyone who grew up watching Star Trek explorers scanning alien worlds with a tricorder. But, it represents an important advance in extraterrestrial research, in which biology has often lagged behind chemistry and geology. Imagine, for instance, virtual-reality headsets that feed mission scientists real-time data as they scan a surface, using a rover’s ‘eyes’ to direct their activities. “To have our team make one of these first steps towards reliably detecting biosignatures using AI is exciting,” she says. “It’s really a momentous time.” More