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    The impact of 1.5 °C and 2.0 °C global warming on global maize production and trade

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    High deforestation trajectories in Cambodia slowly transformed through economic land concession restrictions and strategic execution of REDD+ protected areas

    Deforestation trajectories and economic driversCambodia has undergone significant forest loss in recent decades—with 2.6 million hectares of forest cover loss occurring since 2001, equating to 29.5% of forest cover7 and 1.45 billion tonnes of CO2 emissions8. The deforestation rates have increased by 76% in the last decade (2011–2021) compared to the previous (2001–2010; Fig. 1b)7. We find forest loss has occurred within three distinct Phases demonstrated by changepoint analysis: (1) Phase 1: steady rise from 2000 to 2009 (average = 0.82%/year), (2) Phase 2: peak years from 2010 to 2013 (average = 2.3%/year), (3) Phase 3: moderate phase from 2014 to 2021 (average = 1.6%/year). Whilst the annual rate of deforestation has declined since the Phase 2, Cambodia currently has the highest country-level annual rate of forest loss globally7, illustrating the relentless deforestation spreading across the landscape. Critically, much of this forest loss and degradation is occurring in mature primary forests (Fig. 1b), which hold significant carbon and are home to rich biodiversity and keystone species17,18,19.
    This deforestation in Cambodia has been attributed to the widespread development of Economic Land Concessions (ELCs), the expansion of numerous agricultural frontiers and relentless illegal logging20,21,22. These drivers have been abetted by the establishment of an extensive national road network (Fig. 1a)20—developed to promote economic growth and urban–rural connectivity23. The majority (88.4%) of these roads have been funded by foreign governments (the People’s Republic of China: 38.5%, Japan: 37.9%, and Republic of Korea: 12.0%)18—all of whom have established land concessions within Cambodia’s borders24 through the allocation of state land into private land for long-term industrial plantations22,25. The expansion of ELCs across Cambodia (average addition of 105,000 ha/year of ELC land since 1998) has been directly attributed to up to 40% of the country’s deforestation21, with further indirect impacts due to encroachment into rural community lands (indigenous areas, community forests, subsistence agricultural fields). This results in landlessness, poverty, and land conflicts, forcing communities to migrate in search of arable land, further contributing to the growing degradation and destruction of forests22,26,27,28,29.Strategic government interventionProtected areas expanded across Cambodia in 1993 following a royal decree26; the legal details of which were delineated in the 2008 Protected Areas Law, introducing protected categories, wildlife corridors and strict laws prohibiting development9. While over 80 protected areas currently exist covering 35% of Cambodian land10, they are still under substantial threat30. In further efforts to curb deforestation, the Royal Government of Cambodia ordered the suspension of new ELCs and revocation of a subset of existing ELCs in 2012 (Order 01BB)31. This resulted in a reduction of ELCs from a peak of ~ 2.1 million ha in 2012 to ~ 1.6 million ha from 2014 onward (Fig. 1b), with a significant positive correlation between the quantity of land classified as ELCs and the country-level deforestation rate (R = 0.87, p  More

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    Effect of DNA methylation, modified by 5-azaC, on ecophysiological responses of a clonal plant to changing climate

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    Brain de novo transcriptome assembly of a toad species showing polymorphic anti-predatory behavior

    Sample collection and RNA preparationWe analyzed 6 adult yellow-bellied toad individuals representative of distinct behavioral profiles, i.e. prolonged unken-reflex display vs no unken-reflex display (thereafter referred as “ + ” and “-“, respectively). Behavioral profiles were scored as in Chiocchio et al.12: 3 toads showed prolonged unken-reflex (+), whereas the other 3 did not show unken-reflex (−), as reported in Table 1. Sampling procedures were approved by the Italian Ministry of Ecological Transition and the Italian National Institute for Environmental Protection and Research (ISPRA; permit number: 20824, 18-03-2020). After dissection, brain tissue was immediately stored in RNAprotect Tissue Reagent (Quiagen) until RNA extraction. RNA extractions were performed using the RNeasy Plus Kit (Quiagen), according to the manufacturer’ instructions. RNA quality and concentration were assessed by means of both a spectrophotometer and a Bioanalyzer (Agilent Cary60 UV-vis and Agilent 2100, respectively – Agilent Technologies, Santa Clara, USA).Table 1 Summary of the 6 libraries deposited in the Sequence Read Archive (SRA) of NCBI, in terms of number of raw and trimmed reads per sample.Full size tableLibrary preparation and sequencingLibrary preparation and RNA sequencing were performed by NOVOGENE (UK) COMPANY LIMITED using Illumina NovaSeq platform. Library construction was carried out using the NEBNext® Ultra ™ RNA Library Prep Kit for Illumina®, following manufacturer instructions. Briefly, after the quality control check, the mRNA sample was isolated from the total RNA by using magnetic beads made of oligos d(T)25 (i.e. polyA-tail mRNA enrichment). Subsequently, mRNA was randomly fragmented, and a cDNA synthesis step proceeded using random hexamers and the reverse transcriptase enzyme. Once the synthesis of the first chain has finished, the second chain was synthesized with the addition of the Illumina buffer, dNTPs, RNase H and polymerase I of E.coli, by means of the Nick translation method. Then, the resulting products went through purification, repair, A-tailing and adapter ligation. Fragments of the appropriate size were enriched by PCR, the indexed P5 and P7 primers were introduced, and the final products were purified. Finally, the Illumina Novaseq 6000 sequencing system was used to sequence the libraries, through a paired-end 150 bp (PE150) strategy. We obtained on average 52.7 million reads for each library. The sequencing data are available at the NCBI Sequence Read Archive (project ID PRJNA76401320).Pre-assembly processing stageA total of 316,329,573 pairs of reads was generated by Illumina sequencing. All of them went to a cleaning analytic step. The quality of the raw reads was assessed with the FastQC 0.11.5 tool (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc), in order to estimate the RNAseq quality profiles. The quality estimators were generated for both the raw and trimmed data. The quality assessment metrics for trimmed data were aggregated across all samples into a single report for a summary visualization with MultiQC software tool21 v.1.9 (see Fig. 1). To remove low quality bases and adapter sequences, raw reads were also analyzed through a quality trimming step with Trimmomatic22, v.0.39 (setting the option SLIDINGWINDOW: 4: 15, MINLEN: 36, and HEADCROP: 13). All the unpaired reads were discarded. After the cleaning step and removal of low-quality reads, 297,354,405 clean reads (i.e. 94% of raw reads) were maintained for building the de novo transcriptome assembly (see Table 1).Fig. 1The cleaned reads from all samples were assessed with FastQC and visualized with MultiQC. (a) Read count distribution for mean sequence quality. (b) Mean quality scores distribution. (c) Read length distribution. (d) Per Sequence GC Content.Full size image
    De novo transcriptome assembly and quality assessmentAs there is no reference genome for B. pachypus, we performed a de novo transcriptome assembly procedure. The workflow of the bioinformatic pipelines is shown in Fig. 2. All the described bioinformatics analyses were performed on the high-performance computing systems provided by ELIXIR-IT HPC@CINECA23.Fig. 2Workflow of the bioinformatic pipeline, from raw input data to annotated contigs, for the de novo transcriptome assembly of B. pachypus.Full size imageTo construct an optimized de novo transcriptome, avoiding chimeric transcripts, we employed rnaSPAdes24, a tool for de novo transcriptome assembly from RNA-Seq data implemented in the SPAdes v.3.14.1 package. rnaSPAdes automatically detected two k-mer sizes, approximately one third and half of the maximal read length (the two detected k-mer sizes were 45 and 67 nucleotides, respectively). At this stage, a total of 1,118,671 assembled transcripts were generated by rnaSPAdes runs, with an average length of 689.41 bp and an N50 of 1474 bp (Table 2).Table 2 Similarity rate of newly assembled transcripts versus the de novo transcriptome of B. pachypus.Full size tableResults from the assembly procedures were validated through three independent validator algorithms implemented in BUSCO25 v.4.1.4, DETONATE26 v.1.11 and TransRate27 v.1.0.3. These tools generate several metrics used as a guide to evaluate error sources in the assembly process and provide evidence about the quality of the assembled transcriptome. Busco provides a quantitative measure of transcriptome quality and completeness, based on evolutionarily-informed expectations of gene content from the near-universal, ultra-conserved eukaryotic proteins (eukaryota_odb9) database. Detonate (DE novo TranscriptOme rNa-seq Assembly with or without the Truth Evaluation) is a reference-free evaluation method based on a novel probabilistic model that depends only on the assembly and the RNA-Seq reads used to construct it. Transrate generates standard metrics and remapping statistics. No reference protein sequences were used for the assessment with Transrate. The main metrics resulted from the assembly validators are shown in Table 2 (“Before CD-HIT-est” column). After this triple assessment validation step, the result of the assembly procedure become the input for the CD-HIT-est v.4.8.128 program, a hierarchical clustering tool used to avoid redundant transcripts and fragmented assemblies common in the process of de novo assembly, providing unique genes. CD-HIT-est was run using the default parameters, corresponding to a similarity of 95%. Subsequently, a second validation step was launched on the CD-HIT-est output file. To refine the final transcriptome dataset, a further hierarchical clustering step was performed by running CORSET v1.0629. Then, the output of CORSET was validated by BUSCO, and quality assessment was performed with HISAT230,31 by mapping the trimmed reads to the reference transcriptome (unigenes). Results from all validation steps are shown in Table 2 and discussed in the “Technical Validation” paragraph.Finally, the CORSET output was run on TransDecoder32,33, the current standard tool that identifies long open read frames (ORFs) in assembled transcripts, using default parameters. TransDecoder by default performs ORF prediction on both strands of assembled transcripts regardless of the sequenced library. It also ranks ORFs based on their completeness, and determines if the 5 ‘end is incomplete by looking for any length of AA codons upstream of a start codon (M) without a stop codon. We adopted the “Longest ORF” rule and selected the highest 5 AUG (relative to the inframe stop codon) as the translation start site.Transcriptome annotationWe employed different kinds of annotations for the de novo assembly. We introduced DIAMOND34, an open-source algorithm based on double indexing that is 20,000 times faster than BLASTX on short reads and has a similar degree of sensitivity. Like BLASTX, DIAMOND attempts to determine exhaustively all significant alignments for a given query. Most sequence comparison programs, including BLASTX, follow the seed-and-extend paradigm. In this two-phase approach, users search first for matches of seeds (short stretches of the query sequence) in the reference database, and this is followed by an ‘extend’ phase that aims to compute a full alignment. The following parameter settings were applied: DIAMOND-fast DIAMOND BLASTX-t 48 -k 250 -min-score 40; DIAMOND-sensitive: DIAMOND BLASTX -t 48 -k 250 -sensitive -min-score 40.Contigs were aligned with DIAMOND on Nr, SwissProt and TrEMBL to retrieve the corresponding best annotations. An annotation matrix was then generated by selecting the best hit for each database. Following the analysis of BLASTX against Nr, SwissProt and TremBL, we obtained respectively: 123,086 (64.57%), 77,736 (40.78%), 122,907 (64.48%) contigs. The results obtained following the analysis with BLASTP against Nr, SwissProt and TrEMBL were 96,321 (50.53%), 57,877 (30.36%) and 97,256 (51.02%) contigs respectively. All the information on the resulting datasets is resumed in Table 3.Table 3 Summary of homology annotation hits on the different databases queried in this study.Full size tableThe output obtained by the BLASTX annotation consisted in a total of 77391 sequences simultaneously mapped on the three queried databases (i.e., Nr, SwissProt and TrEMBL). The output obtained following the BLASTP annotation consisted in a total of 57704 sequences simultaneously mapped on the three databases. Venn diagrams are presented in Fig. 3, showing the redundancy of the annotations in the different databases for both DIAMOND BLASTX (Fig. 3a) and DIAMOND BLASTP (Fig. 3b). Furthermore, the ten most represented species and the ten hits of the gene product obtained respectively with BLASTX and BLASTP by mapping the transcripts against the reference database Nr are shown in Figs. 4 and 5. Since BLASTX translated nucleotide sequence searches against protein sequences the BLASTX results are more exhaustive than BLASTP results. Contigs were also processed with InterProScan35 to detect InterProScan signatures. The InterPro database (http://www.ebi.ac.uk/interpro/) integrates together predictive models or ‘signatures’ representing protein domains, families and functional sites from multiple, diverse source databases: Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY and TIGRFAMs. The obtained InterProScan results for all the unigenes are available on Figshare in the form of Tab Separated Values (tsv) file format, which includes the GO and KEGG annotated contigs, respectively.Fig. 3Venn diagrams for the number of contigs annotated with DIAMOND (BLASTX (a) and BLASTP (b) functions) against the three databases: Nr, SwissProt, TREMBL.Full size imageFig. 4Most represented species and gene product hits. Top 10 best species (a) and protein (b) hits present in the reference database (Nr, BLASTX).Full size imageFig. 5Most represented species and gene product hits. Top 10 best species (a) and protein (b) hits present in the reference database (Nr, BLASTP).Full size imageComparison with Bombina orientalis brain transcriptomeWe compared the brain de novo transcriptome of B. pachypus with the brain de novo transcriptome of B. orientalis, recently produced in the frame of a prey-catching conditioning experiment17,18. The B. orientalis transcriptome resource was downloaded from GEO archive of NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171766). To make the datasets comparable, we first performed ORF prediction on B. orientalis trascriptome using Transdecoder, using default settings. Results from the B. orientalis trascriptome ORF prediction are available in Figshare at the following link https://doi.org/10.6084/m9.figshare.20319633/). We also applied the makedb function implemented in DIAMOND to create the protein database index. Then, we aligned the B. pachypus predicted coding sequences and proteins (query files) against the B. orientalis protein database (reference) using DIAMOND BLASTX and BLASTP, respectively. We obtained 167041 matches from BLASTX and 156248 matches for BLASTP. Results from the BLASTX and BLASTP comparisons, and the most matched proteins, are available on Figshare36 (link available in next paragraph). More

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    Low functional vulnerability of fish assemblages to coral loss in Southwestern Atlantic marginal reefs

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    To compare soil viral community composition within and across terrestrial habitats on a regional scale, viromes were generated from 34 near-surface (top 15 cm) soil samples, with a total of 30 viromes included in downstream ecological analyses (see Supplementary Methods). The analyzed viromes were collected from four distinct habitats (wetlands, grasslands, chaparral shrublands, and woodlands, each with 7, 14, 4, and 5 viromes, respectively) across five field sites (Fig. S1 for sampling scheme, Table S1 for soil properties). Following quality filtering, the 30 viromes generated an average of 72,950,833 reads and 416 contigs ≥10 Kbp per virome (Table S2). Wetland viromes yielded more contigs ≥10 Kbp than viromes from other habitats, both in total and on average per virome (Table S2). We used VIBRANT to identify 3490 viral contigs in our assemblies, which were clustered into 3,432 viral operational taxonomic units (vOTUs), defined as ≥10 Kbp viral contigs sharing ≥ 95% average nucleotide identity over 85% contig length [17]. Constrained analysis of principal coordinates (CAP analysis) revealed strong clustering by habitat rather than by site, implying that, where environmental parameters are substantially different, environmental conditions are stronger drivers of viral community composition than geographic distance (Fig. S2).Multiple lines of evidence suggest that wetter soils harbored greater viral diversity than drier soils. We recovered the most vOTUs from wetlands, both in total (56% of all vOTUs were from wetlands) and per virome (on average, 307 vOTUs were recovered per wetland virome, compared to 116 from all habitats) (Fig. 1A). Unsurprisingly, wetlands had significantly greater moisture content than other habitats (Fig. 1B; ANOVA followed by Tukey multiple comparisons of means, p 100 Km distances here. Taken together, we propose that soil viral communities often display high heterogeneity within and among habitats, presumably due to a combination of host adaptations and microdiversity, dispersal limitation, and fluctuating environmental conditions over space and time. More

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