Philippa Kaur

Animals and dietsThe animal experiments were conducted in accordance with the Guidelines for Animal Experiments and Act on Welfare and Management of Animals, Hokkaido University, and all experimental procedures were approved by the Animal Care and Use Committee of Hokkaido University. All animal experiments were carried out in accordance with ARRIVE guidelines. Twenty newborn female Holstein calves with an average birth weight of 37.1 ± 1.0 kg (mean ± standard error) were randomly allocated to either the control or treatment group at birth. All calves were housed individually in separate calf hutches containing sawdust bedding. Feeding and managing of animals until weaning at 50 d of age was performed as described previously17. After supplementing colostrum at birth, calves in both groups were fed 4 L of pasteurized whole milk (44.2% crude protein [CP] and 29.3% fat on a dry matter [DM] basis) as a transition milk during the first week since birth. From 8 days until weaning at 50 days of age, milk replacer (28.0% CP and 18.0% fat on a DM basis) was fed twice daily at 0830 and 1600 h. Water, calf starter (22.9% CP, 11.0% neutral detergent fiber [NDF], 5.6% acid detergent fiber [ADF], 6.2% crude ash, and 3.0% ether extract on a DM basis), and chopped Timothy hay (3.4% CP, 53.1% NDF, 34.2% ADF, 4.3% crude ash, and 1.7% ether extract on a DM basis) were provided for ad libitum intake from 3 days of age. In addition to voluntary intake of solid diets, the calves in the treatment group were orally administered with a mixture of ground Timothy hay and psyllium (4.4% CP, 78.6% NDF, 5.8% ADF, 3.9% crude ash, and 0.3% ether extract on a DM basis) from 3 days until weaning at 50 days of age. Timothy hay was ground for oral administration using a Wiley grinder (WM-3, Irie Shokai) with a 2-mm screen. To improve the handling of the treatment diet for oral administration, we incorporated psyllium, which is a dietary fiber that primarily improves gastrointestinal conditions in humans and can be incorporated in oral electrolyte solution supplemented to neonatal calves38. As a treatment diet, ground Timothy hay (50 g) and psyllium (6 g) were mixed with 200 mL of water. Owing to the adhesiveness of psyllium, the treatment diet formed a “hay ball” and showed slight stickiness, which facilitates swallowing by calves. At 3–7 days of age, one hay ball (50 g of fibrous diet) was orally administered after morning milk feeding. From 8 days of age to weaning, an additional hay ball was fed immediately after evening milk feeding (100 g fibrous diet per day).After weaning, animals in both dietary groups were merged into the same herd and managed on the same farm under identical conditions. From 9 months of age until calving, heifers were fed a ration containing Timothy hay, alfalfa hay, fescue hay, and concentrate. After calving, the cows were fed a diet for lactating cows, as described in Supplementary Table S8. Diets comprised a total mixed ration and were fed twice daily at 0900 and 1600 h. All animals had ad libitum access to water and mineral blocks throughout the experiment. Daily milk production for each cow was measured for the first 30 days of the lactation period and the average values for each dietary group on a weekly and monthly basis were calculated. Milk yield for four animals in each dietary group were not recorded due to health problems including mastitis and displaced abomasum symptoms after calving.In this study, all animals (n = 20) were maintained until 9 months of age, without severe problems. Owing to health problems, several animals were excluded from the experiment before parturition as follows: three animals (one in the control group and two in the treatment group) at 60 days before the expected calving date and one animal in the control group at 21 days before the expected calving date. One animal in the control group (15 days after calving) and two animals in the treatment group (calving day) were diagnosed with displaced abomasum symptoms and were excluded from further sampling. Owing to technical problems, samples were not collected from three animals aged 7 days in the treatment group and one animal aged 21 days in the control group. All other samples (n = 176) were obtained at the target sampling points.Sampling of rumen contentsRumen contents were collected orally using a stomach tube. The stomach tube and the sample collection flask were thoroughly cleaned using water between sample collections from individual animals; the first fraction of the sample was discarded to avoid contamination from the previous sample and saliva. All samples were collected at 4 h after morning feeding. Rumen contents were collected at 7, 21, 35, 49, and 56 days, and at 9 months of age, 60 and 21 days before the expected calving date, at calving day, and 21 days after calving. The pH was measured using a pH meter (pH meter F-51; Horiba, Kyoto, Japan) immediately after sampling. Samples were collected in a sterile 50 mL tube and immediately placed on ice, followed by storage at − 30 °C until use.Chemical analysisRumen contents (1.0 g) were centrifuged at 16,000×g at 4 °C for 5 min, and the supernatant was collected. The SCFA content was analyzed using a gas chromatograph (GC-14B; Shimadzu, Kyoto, Japan) as described previously39. In brief, the supernatant of the rumen contents was mixed with 25% meta-phosphoric acid at a 5:1 ratio, incubated overnight at 4 °C, and centrifuged at 10,000×g at 4 °C. The supernatant was then mixed with crotonic acid as an internal standard and injected into a gas chromatograph equipped with an ULBON HR-20 M fused silica capillary column (0.53 mm i.d. × 30 m length, 3.0 µm film; Shinwa, Kyoto, Japan) and a flame-ionization detector. d/l-lactic acid levels were measured using a commercial assay kit (Megazyme International Ireland, Wicklow, Ireland) according to the manufacturer’s instructions. NH3-N levels were measured via the phenol-hypochloride reaction method40 using a microplate reader at 660 nm (ARVO MX; Perkin Elmer, Yokohama, Japan).DNA extraction and rumen microbiota profiling via amplicon sequencingTotal DNA was extracted and purified using the repeated bead-beating plus column method41. Rumen contents (0.25 g) were homogenized using sterile glass beads (0.4 g; 0.3 g of 0.1 mm and 0.1 g of 0.5 mm) and cell lysis buffer (1 mL; 500 mM NaCl, 50 mM Tris–HCl [pH 8.0], 50 mM ethylenediaminetetraacetic acid (EDTA), and 4% sodium dodecyl sulfate). The lysates were then incubated at 70 °C for 15 min, and the supernatant was collected for further processing. Bead-beating and incubation steps were repeated once, and all supernatants were combined. Total DNA was precipitated using 10 M ammonium acetate and isopropanol, followed by purification using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). The DNA concentration was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and adjusted with Tris–EDTA buffer to the appropriate concentration.For a comprehensive analysis of rumen bacterial communities, the MiSeq sequencing platform (Illumina, San Diego, CA, USA) was used. Total DNA obtained from the rumen contents was diluted to a final concentration of 5 ng/μL and subjected to PCR amplification of the V3-V4 regions of the 16S rRNA gene using the primer sets S-D-Bact-0341-b-S-17 (5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′-GACTACHVGGGTATCTAATCC-3′)42. The PCR mixture consisted of 12.5 μL of 2× KAPA HiFi HotStart Ready Mix (Roche Sequencing, Basel, Switzerland), 0.1 μM of each primer, and 2.5 μL of DNA (5 ng/μL). PCR amplification was performed according to the following program described previously9: initial denaturation at 95 °C for 3 min; 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final extension step at 72 °C for 5 min. Amplicons were purified using AMPure XP beads (Beckman-Coulter, Brea, CA, USA) and subjected to sequencing on the Illumina MiSeq platform (Illumina) using the MiSeq Reagent Kit v3 (2 × 300 paired-end). Data obtained from amplicon sequencing using the MiSeq platform were analyzed using QIIME2 version 2019.443. Paired reads were filtered, dereplicated, merged, and chimera-filtered using the q2-dada2 plugin44 to generate ASVs. Taxonomic classification of the ASVs was performed at the phylum, class, order, family, and genus levels using the SILVA 132 99% operational taxonomic units, full length, seven level taxonomy classifier (silva-132-99-nb-classifier.qza). Sequenced data were processed further and analyzed using R software version 3.6.245. ASV and taxonomy tables generated using QIIME2 were imported into R and merged with the sample metadata using the Phyloseq Bioconductor packages46. ASVs identified as Archaea, chloroplasts, and mitochondria were excluded. All samples were rarefied to a sampling depth of 16,805 reads, which was the smallest number of reads observed per sample in the filtered ASV table. Alpha diversity indices including Chao1, ACE, Shannon, and Simpson indices were calculated using the phyloseq function “estimate_richness”. PCoA was performed to determine differences in the microbial community structure based on the Bray–Curtis dissimilarity matrices at the genus level using the Phyloseq package. Venn diagrams were generated using ASVs showing mean relative sequence abundances of  > 0.1% in either the control or the treatment groups at each sampling point. The relative abundance of each bacterial taxon was calculated by dividing the number of reads assigned to each taxon by the total number of reads. Taxa with an average relative abundance  > 0.1% in  > 50% of samples in either the control or treatment group during at least one sampling point were used for the analysis. Hierarchical cluster analysis of bacterial genera determined via amplicon sequencing at 21 days after calving and the weekly and monthly average milk yield for the first 30 days of lactation period was performed using the distances calculated from Spearman’s correlation and average linkage clustering.Quantification of target bacterial species/groups using real-time PCRThe relative abundance of known ruminal bacterial species and groups, including the total bacteria, F. succinogenes, R. flavefaciens, Ruminococcus albus, Butyrivibrio spp., Prevotella spp., Selenomonas ruminantium, Megasphaera elsdenii, Treponema spp., Streptococcus bovis, Anaerovibrio lipolytica, and Ruminobacter amylophilus, was quantified using real-time PCR. Amplification was performed using a Light Cycler 480 system (Roche Applied Science, Mannheim, Germany) with a KAPA SYBR Fast qPCR Kit (Roche Sequencing, Basel, Switzerland) and the respective primer sets (Supplementary Table S9). The standards used for the real-time PCR were prepared as described previously47. Briefly, plasmid DNA containing the respective target bacterial 16S rRNA gene sequence was obtained by PCR cloning using the species/genus-specific or bacterial universal primer sets. The concentration of the plasmid was determined with a spectrometer. Copy number of each standard plasmid was calculated using the molecular weight of nucleic acid and the length (base pair) of the cloned standard plasmid. Ten-fold dilution series ranging from 1 to 108 copies were prepared for each target and run along with the samples. The respective genes were quantified using standard curves obtained from the amplification profile of the dilution series of the plasmid DNA standard (Supplementary Table S9). The PCR cycling conditions and reaction mixture were the same as those reported previously48. The relative abundance of each bacterial target was expressed as the proportion (%) of the abundance of the 16S rRNA genes of each bacterial target relative to that of the total bacteria.Statistical analysisAll data were sorted based on animal age into two sets, from 7 to 56 days of age and from 9 months of age to 21 days after calving, and analyzed separately. Data on fermentation parameters and bacterial abundance quantified via real-time PCR were analyzed using a repeated measures model using GraphPad Prism software version 9.1 (GraphPad Software, San Diego, CA, USA) with the fixed effects of dietary group, age, and diet × age interaction, and the random effect of animals within the groups. The Greenhouse–Geisser correction was used where sphericity was violated. If the P-value for the treatment effect was  More

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Proteobacteria are major contributors to marine microbial phosphonate cyclingDatabases for all putative sequences of genes for phosphonate production (pepM, aepY, phpC, mpnS, hepD), substrate-specific catabolism (phnAWXYZ, palA), and broad-specificity catabolism (phnIJM) were created using available public genomes from JGI IMG/MER and GORG-Tropics. Gene identity was verified by the presence of catalytically essential residues (Supplementary Table S2). Phosphonate genes were identified in 10,337 genomes of bacteria and archaea spanning over 100 unique classes, suggesting a wide variety of microorganisms mediate phosphonate production and catabolism (Fig. 2, Supplementary Dataset S1). A high proportion of all collected sequences affiliated with Proteobacteria (Gamma, Alpha, and Beta classes), averaging 52% of the production genes, 78% of substrate-specific catabolism genes, and 88% of broad-specificity catabolism genes before dereplication (Fig. 2).Fig. 2: Phosphonate gene and genome count with taxonomic distribution.Number of sequences and genomes collected for study (A, D, G) with distribution of class-level taxa for all redundant sequences (B, E, H) and marine redundant (C, F, I) sequences. Results are shown for selected genes representing phosphonate (A–C) production, (D–F) substrate-specific catabolism, and (G–I) broad-specificity catabolism. The taxa shown are the 15 classes with the highest representation across all databases.Full size imageOf the 10,337 genomes, 1556 (15%) were confirmed to be marine organisms from 35 different classes (Fig. 2, Supplementary Dataset S1). Proteobacteria had even greater representation in the subset of marine genomes, averaging 65% of marine production genes, 88% of marine substrate-specific catabolism genes, and 96% of marine broad-specificity catabolism genes from the redundant databases (Fig. 2). The dominance of Alphaproteobacteria in the marine subset may be attributed to the wide variety of Pelagibacterales bacterium captured in the database, making up 426 (27%) of the 1556 genomes involved in all three categories of phosphonate cycling. Rhodobacterales (Ascidiaceihabitans sp., Roseovarius sp., Sulfitobacter sp., Labrenzia sp., and Phaeobacter sp.) alongside Rhodospirillales (Thalassobaculum sp., Thalassospira sp., Roseospira sp., Varunaivibrio sp., and Oceanibaculum sp.) were also highly represented among the marine subset with 214 (14%) and 251 (16%) genomes, respectively (Supplemental Dataset S1), though these taxa primarily show potential for phosphonate catabolism rather than production. Vibrionales were well represented in the JGI IMG/MER marine genome subset with 107 (7%) genomes spanning 59 different species including Vibrio lentus, Vibrio breoganii, and Vibrio splendidus.Diverse taxa encode the capacity to produce phosphonate derivativesPhosphonate production is widespread and distributed throughout many different bacteria and archaea. Genes responsible for the first two steps in phosphonate production, pepM and aepY, had the broadest taxonomic distribution within the redundant databases (Shannon indices of 2.66 and 2.76) for all genes in this study, distributed with 0.59 and 0.61 evenness from 70 and 72 unique, verified classes, respectively. Their broad distribution further highlights the ubiquity and necessity of phosphonate compounds to microbial life and function across all environments. Within the marine setting, both pepM and aepY have reference sequences from 22 unique, verified classes which is the second highest class representation in the marine genome subset (Fig. 2). The marine subset of pepM and aepY also have the highest Shannon indices (1.76 and 1.92) distributed with 0.53 and 0.58 evenness, respectively. A majority (87%) of the Alphaproteobacteria phosphonate producers are Pelagibacterales bacterium with other notable taxa including Bacteria: Candidatus Actinomarinaceae, Prochlorococcus sp., Synechococcus sp., Nitrosococcus sp., and MG-I Archaea: Candidatus Nitrosomarinus catalina, Nitrosopumilus maritimus, alongside other unidentified Crenarchaeota and Thaumarchaeota genomes.The gene phpC was found in less than half the number of genomes than pepM and aepY, and encoded by fewer classes in both the general database (47) and marine subset (10). In the full databases, the distribution of retrieved phpC sequences are similar to pepM and aepY with respect to taxonomic ranking, Shannon index (2.49), and evenness (0.60) (Fig. 2A–C, Supplementary Table 5). Within the marine subset, phpC has less Shannon index (1.61) but greater evenness (0.61) than the marine subset of pepM and aepY. All three upstream phosphonate production genes (pepM, aepY, phpC) are found together within Pelagibacterales bacterium, Prochlorococcus sp., Thaumarchaeota, and Crenarchaoeta alongside other taxa such as Oceanospirillales sp., Arenimonas donghaensis, Desulfuromusa kysingii, and Cellulosilyticum lentocellum.We further investigated the relationship between pepM, aepY, and phpC by examining co-occurrence in genomes and synteny with the general, redundant databases. The first two steps in phosphonate biosynthesis are intimately linked (Fig. 3). Out of all genomes with pepM, 86% have aepY, and out of all genomes with aepY, 90% have pepM. By contrast, phpC is not as closely tied to pepM and phosphonate production. We found phpC in just over 20% of genomes with the capability of phosphonate production (Fig. 3), implying that a majority of bacterial and archaeal phosphonate production stops at the production of phosphonoacetaldehyde or 2-AEP (Fig. 1A). Furthermore, half of the phpC genes were not associated with phosphonate production, given 53% of genomes with phpC did not have pepM and 54% did not have aepY (Fig. 3). In these instances, microbes may use phpC within a 2-AEP substrate-specific catabolism operon (Fig. 3) that allows phosphonate compounds to be synthesized by transforming 2-AEP with phnW and phpC into 2-HEP (Figs. 1A and 3). By repurposing 2-AEP, individuals can still create the specific compound needed while bypassing the energetically unfavourable first step of phosphonate production.Fig. 3: Co-occurrence of phosphonate cycling genes within the same genome and examples of genetic organization of phosphonate cycling genes.The heatmap displays co-occurrence of phosphonate cycling genes. Each column represents the subset of all genomes which contain the source gene and the heatmap value represents the fraction of the source genomes which also contain the co-occurring gene. Heatmap values are not symmetrical due to differing number of genomes represented in each column, database size listed above each column. Examples for phosphonate cycling genomic neighbourhoods were chosen to maximize diversity in synteny with examples from both Bacteria and Archaea where applicable. Several phosphonate-specific ABC transport system clusters are labelled as follows: phnC = phosphonate transport system ATPase; phnD = phosphonate transport system substrate-binding; phnE = phosphonate transport system permease; phnS = 2-AEP transport system substrate-binding; phnT = 2-AEP transport system ATP-binding; phnV = 2-AEP transport system permease; palC = transport system permease; palD = transport system ATP-binding; palE = transport system permease. Genes are colour coded by: red = lyase; orange = transcriptional regulator; yellow = hydrolase; green = transferase; light blue = oxidoreductase; dark blue = transaminase; purple = kinase; pink = isomerase; brown = transport; white = synthase; black = uncharacterized protein; grey = unknown.Full size imageA narrow but diverse selection of taxa encoded MpnS, the marker gene for Mpn production and a key determinant in marine methane production. We observed distinct clades of this enzyme in autotrophic archaea and heterotrophic bacteria (Fig. 2B, C). Within the marine ecosystems, Pelagibacterales, Rhodospirillales, Rickettsiales, Oceanospirillales, Flavobacteriales, and Synechococcales are bacterial candidates for MPn production alongside Thaumarchaeota and Crenarchaeota archaeon (Fig. 2B, C). While six of the bacterial genomes with MpnS also encoded genes for phosphonate catabolism, none of the archaeal MPn producers showed capacity for catabolism (Supplementary Dataset S1). The genomic neighbourhoods for general phosphonate production (pepM, aepY, phpC) and MPn production (mpnS) in both bacteria and archaea include genes such as glycosyltransferase, lipopolysaccharide choline phosphotransferase, choline kinase, adenylyltransferase, and arylsulfatase A (Fig. 3) suggesting the potential for synthesis of (methyl)phosphonate esters [93]. This is consistent with previous analysis [29] of the Nitrosopumilus maritimus SCM1 MPn production genomic neighbourhood and biophysical evidence that MPn producing archaea synthesize an exopolysaccharide modified with MPn similar to 2-AEP modified polymers.Contrary to the diversity of the other phosphonate production databases, the hepD database has low Shannon index (0.62) and evenness (0.45) with 79% of sequences mapping to Actinomycetia including Streptomycetales and Corynebacteriales (Fig. 2B, C). The marine subset has lower Shannon index (0.28) and evenness (0.41) where all sequences derive from Pelagibacterales except one from Prochlorococcus sp. The genomic neighbourhood of HMP production may contain genes for cell surface modification such as acetyltransferase, peptidoglycan biosynthesis, and adenylylsulfate transferase, suggesting that some organisms may use HMP as a conjugate for membrane-associated or exported macromolecules similar to theories on MPn utilization. Other examples of hepD synteny contain more specific genes such as the HMP dehydrogenase or other enzymes for downstream modification (Fig. 3).Marine proteobacteria encode genes for substrate-specific and broad-specificity phosphonate catabolismGenes for marine substrate-specific phosphonate catabolism were widespread among Proteobacterial classes, and to a lesser extent amongst other classes including Bacilli, Planctomycetes, and Synechococcus (Fig. 2E,F). Marine substrate-specific catabolism has lower average Shannon index (1.00) and evenness (0.43) than the three general production genes (pepM, aepY, phpC). The most widespread of these genes was phnW, likely due to its pivotal role in 2-AEP transformations as a precursor reaction to phnAY or phnX (Fig. 1, Supplementary Table 5). Marine hydrolases for 2-AEP catabolism, phnA, phnX, and phnZ, have similar Shannon indices (mean: 1.11 ± 0.05) and evenness (mean: 0.41 ± 0.03) (Fig. 2E, F, Supplementary Table 5).While not exclusive, sequenced references demonstrate a strong taxonomic partition between Proteobacterial classes for 2-AEP catabolism pathways phnAWY and phnWX. Over 74% of marine genomes with phnAWY are Alphaproteobacteria, in particular Rhodobacterales species such as Roseovarius nubinhibens, Marivita geojedonensis, and Pelagicola litoralis. On the contrary, ~80% of marine genomes with phnWX are Gammaproteobacteria, specifically of Vibrionales, Oceanospirillales, and Alteromonadales including a wide range of species from Vibrio, Photobacterium, Marinobacterium, Halomonas, and Pseudoalteromonas.Taxonomic distribution for marine phnZ was 72% Alphaproteobacteria with Pelagibacterales making up 45% of marine phnZ sequences. Note that phnZ has the most (17) reference sequences from marine Cyanobacteriia, specifically Prochlorococcus sp., than any other phosphonate catabolizing gene. Lack of marine sequence representatives for catabolism of phosphonopyruvate by palA suggests that either the substrate is uncommon, therefore the function unnecessary, or marine microbes have other methods of catabolizing phosphonopyruvate, perhaps by the C-P lyase. Overall taxonomic distribution of phosphonate substrate-specific catabolism, specifically targeting 2-AEP, suggests said function is essential to many marine heterotrophs within Alphaproteobacteria and Gammaproteobacteria. However 2-AEP catabolism appears to be less universally important than phosphonate production to marine microbial life since the required genes are found in a less diverse selection of taxa.Genetic organization for substrate-specific catabolism genes, particularly those targeting 2-AEP, varied widely in line with the numerous options for 2-AEP catabolism (Fig. 3). Though some bacteria specialize in a single 2-AEP degradation pathway such as only containing phnWAY, others contained multiple hydrolases for 2-AEP catabolism with some incorporating phpC into a 2-AEP specific catabolism operon (Fig. 3). When a genome has two hydrolases for phosphonate catabolism, often phnZ was paired with either phnA or phnX. Co-occurrence between phnZ and either phnA or phnX ranged between 30-50%, whereas co-occurrence between phnA and phnX was between 6-12% (Fig. 3). This discrepancy in co-occurrence may be due to the metabolic similarity between phnA and phnX, where having both may be redundant. Both of these enzymes rely on phnW for 2-AEP catabolism and produce carbon metabolites, whereas phnZ does not need phnW and produces the amino acid glycine (Fig. 1B).C-P lyase genes representing substrate non-specific catabolism were overwhelmingly attributed to Alphaproteobacteria which consisted over 75% of all collected marine sequences for phnIJM (Fig. 2H, I). A wide variety of Rhodobacterales, spanning 55 different genus are the most numerous representatives, followed by Pelagibacterales and Rhodospirillales. The genes in all three databases have very high genome co-occurrence, 89–99%, as expected given all three operate within the same enzyme complex (Fig. 3). Gene co-occurrence, Shannon index, and evenness is lower for phnM than the other two C-P lyase components, phnI and phnJ, likely due to instances of organisms containing two copies of phnM where one copy lies outside the C-P lyase operon [94]. C-P lyase gene databases have lower Shannon index (mean 0.81 ± 0.11) than phosphonate production and 2-AEP substrate-specific catabolism genes (phnAWXZ) (Fig. 3D, G), suggesting broad-specificity phosphonate catabolism by the C-P lyase is a narrowly distributed function (Supplementary Table 5). Organization of C-P lyase operons held the most consistency between example genomes, likely due to the high number of genes simultaneously utilized for lyase construction. These operons encoded a consecutive string of lyase subunits, including a generic phosphonate transporter (phnCDE) and GntR transcriptional regulator (Fig. 3). C-P lyase genes had low genomic co-occurrence with all other phosphonate cycling genes with notable co-occurrence between phnW at 26%, phnZ at 21%, and phnX and 18% (Fig. 3). The low rate of co-occurrence may be due to redundancy in function for P harvesting between the C-P lyase and substrate-specific catabolism. In some cases there are instances of a substrate-specific hydrolase gene located within the C-P lyase operon (Fig. 3).Phosphonate biosynthesis genes are globally prevalent in oceans and increase in mesopelagic watersFollowing curation of phn-gene databases, we analysed 121 metagenomes and 91 metatranscriptomes from the publicly available TARA Oceans expedition (spanning samples from the Atlantic, Indian, Pacific, and Southern Ocean and Red Sea) to investigate the global potential for marine phosphonate cycling. Measuring the proportion of the community capable of performing specific tasks through metagenomics indicates the long-term selective pressures that shape P-cycling and microbial communities.Potential for phosphonate production (pepM) was globally ubiquitous across all depths, with 14–17% of the community encoding in the surface waters and deep chlorophyll maximum (DCM), increasing to 45% in mesopalagic waters (Fig. 4A, Supplementary Tables S6 and S7), highlighting the importance of phosphonate compounds to marine microbial communities. Relative abundance of phpC was 64–76% that of pepM and aepY across all depths (Fig. 4A). We observed significant increase in relative abundance between the surface and mesopelagic for pepM (ANOVA: F = 1262, p  More

In their original description of V. rhodanica, Fischer & Riou16 determined that the previously undescribed genus was a Jurassic relative of V. infernalis. This assignment was based on the configuration of the arm crown and armature, fin type, presence of luminous organs, lateral eyes, and the absence of an ink sac. Assuming this assignment is correct, then V. rhodanica is a member of the suborder Vampyromorphina, which includes the family Vampyroteuthidae22,29.Reappraisal of the anatomy shows that V. rhodanica and V. infernalis both have 8 arms and uniserial suckers flanked by cirri. They both possess V. infernalis-like sucker attachments34,36, which are broader at the base and taper up to a radially symmetrical sucker.Both species have distinctly modified arms though the morphology differs in each. V. infernalis, has retractable filaments in the position of arm pair II27,33,34, though there is no evidence of these appendages in V. rhodanica. Instead, the species has elongate dorsal arms (arm pair I) with a unique configuration of suckers and cirri on the distal section.The suckers and cirri of V. rhodanica are more numerous than those of V. infernalis27,37. They are also more closely positioned. Proportionally, the suckers of both species have a consistent ratio to mantle length37, though the diameter of the cirri and infundibulum are greater in V. rhodanica. The V. infernalis-like attachment1,3,34 is present in both species, though in V. rhodanica, the distal part of the neck protrudes into the acetabular cavity. Of note, the sucker stalks on the dorsal arms of V. rhodanica are more elongate than those on the other arms (Figs. 2b,c, and 3a,b). This variation in suckers and their attachments suggests a specialized function between the dorsal and sessile appendages. On the longer dorsal arms, the larger sucker diameter, and more elongate stalks (Figs. 2b and 4) indicate the potential for increased mobility over their extant relatives, and possibly facilitated additional manipulation and prey capture capability.Figure 4Hypothesised reconstruction of V. rhodanica based on the data from this study (A. Lethiers, CR2P). The scale is based on measurements from the holotype (MNHN.B.74247) and the arm crown is completed using dimensions from MNHN.B.74244.Full size imageIn addition to the arm crown specialization, V. rhodanica has a more streamlined shape than V. infernalis, which is caused by a proportionally narrower head. Their muscular body is narrower and more elongate than the gelatinous V. infernalis16,27,37 suggesting a higher energy locomotory style. This is consistent with increased predation relative to the modern form. Observations in this study support many assertions of Fischer & Riou16 about the characters in V. rhodanica, though the presence of luminous organs cannot be confirmed. Rather than luminous organs much larger than those present in the deep-sea, extant V. infernalis, it is possible that these structures represent displaced cartilage prior to fossilization (Supplementary Fig. 6).Two other genera from the La Voulte-sur-Rhône locality, Gramadella and Proteroctopus are, like V. rhodanica, considered to be Incertae sedis Vampyromorpha22. All three share morphological similarities that include an elongated mantle fused with the head, and a longer dorsal arm pair with armature on the distal ends1,16,22,38. Neither the second nor fourth arm pair have been modified. Each has one pair of fins. In Gramadella, the fins are lateral and skirt-like16,38. In V. rhodanica and Proteroctopus these fins are located posteriorly1,16.V. rhodanica shows the greatest length variation between the dorsal and sessile arms (Fig. 4), though proportionally, Gramadella, and Proteroctopus have longer dorsal arms1,31. Fischer & Riou31 and Kruta et al.1 described biserial suckers in their descriptions of Gramadella, and Proteroctopus, respectively. In Proteroctopus, these suckers have a proportionally smaller diameter than the uniserial row in V. rhodanica, and do not exhibit the same tapered pattern.None of these specimens shows evidence of an ink sac, though it is present in contemporaneous genera from the same assemblage (Mastigophora, Rhomboteuthis and Romaniteuthis)8,16. That this character occurs only in some taxa from the same assemblage suggests variation in ecology, possibly associated with the steep, bathymetric relief in the La Voulte-sur-Rhône paleoenvironment11. The mosaic of characters found within the coleoid taxa at La Voulte-sur-Rhône suggests that Mesozoic vampyromorphs co-occurred in different ecological niches during the mid-Jurassic.Today, extant V. infernalis is uniquely adapted to a low-energy, deep-sea mode of life27,28,29,39, though the timing of character acquisition and progression of this ecology is unclear24. It is hypothesised that the vampyromorph Necroteuthis Kretzoi 1942 was already exploiting this niche by the Oligocene29, and that the initial shift to offshore environments was possibly driven by onshore competition24,29. The data obtained here suggests that V. rhodanica, the purportedly oldest-known genus of the Vampyromorphina group, was an active predator following a pelagic mode of life.Indeed, several anatomical details, mainly found in the brachial crown, seem to support this hypothesis. Though we cannot directly compare functionality of the arm crown elements with other Jurassic taxa, we can infer function based on observation in modern forms. In Octopoda, the sister group to Vampyromorpha, suckers are attached to the arm by a cylindrical layer of muscle, encircling oblique musculature40,41, that connects the arm musculature and the lateral margin of the acetabulum34,40,41,42. This facilitates a variety of functions including locomotion, manipulation, and prey retention43. The sucker attaches by flattening the infundibulum against the surface and then the encircling epithelium creates a watertight seal36,40,41,42,43,44,45. Contraction of the radial acetabular muscles provides the pressure differential required to create the suction force43,44,46.The stalked sucker attachments2,34 of decabrachians (Fig. 3d, and Supplementary Fig. 4) are muscular35 and connect the musculature of the arm with the base of the sucker, forming part of the acetabulum33,34. Tension on the sucker stretches this muscular attachment, which pulls locally on the acetabular base. This facilitates a greater pressure differential inside the sucker, allowing the teeth on the sucker ring to maintain the hold47.Extant V. infernalis lack decabrachian-like stalks2,18 and the neck of the attachment joins to the base of the acetabulum (Fig. 3c, and Supplementary Fig. 4), rather than being inserted into it18. The infundibulum is not distinct, and the suckers do not provide strong suction27. Instead, suckers function by secreting mucus to coat detritus—marine snow captured by retractable filaments—which is then moved to the mouth by cirri7,27.A mosaic of these characters is present in V. rhodanica (Fig. 3a,b), therefore, suggesting their potential for increased attachment and hold on prey over extant V. infernalis. These include a larger infundibular diameter, a neck attachment integrated with the acetabular muscles, and the elongated stalks of the dorsal suckers.Additionally, the paired, filamentous cirri observed in extant cirrates48 are present in V. rhodanica (Fig. 4, and Supplementary Fig. 2). In extant forms they are understood to have a sensory function and are used in the detection and capture of prey48. In V. infernalis, they serve to transport the food proximally along the arms to the mouth27. The greater diameters of cirri, and placement along the entire arm in V. rhodanica (Fig. 4), suggests an increased sensory function in these fossil forms.The shape of the arms also contributes to the suction potential49 in coleoids. Functional analysis in Octopoda highlights a positive correlation between distal tapering of the arms and their flexibility. A tapered, flexible arm facilitates more precise adhesion than a cylindrical-shaped one and requires a greater force for sucker detachment49. Suckers detach sequentially, rather than the more simultaneous release observed in models of arms with less taper variation. The tapered diameter of the suckers, like those seen on the sessile arms of V. rhodanica, potentially facilitated this kind of sequential detachment49 allowing them more adherence force and flexibility. Though V. rhodanica has just two suckers on the distal tips of their dorsal arms, the most distal is marginally smaller in diameter than the proximal one. On the dorsal arms, this tapering is observed in conjunction with a well-developed axial nerve cord (Fig. 2b). In extant forms, the nerve cord facilitates complex motor functions42. The combination of these characters in V. rhodanica suggests their arms had increased potential to be actively used in prey capture50 over extant V. infernalis.Though arm crown characters offer insight on the ecology of V. rhodanica, in fossil coleoid phylogenies only a few characters are based on the suckers1, 3. Two studies that have attempted to create a phylogeny using morphological characters that include both fossil and extant taxa return V. rhodanica and V. infernalis as sister taxa1,3. These matrices are, by necessity, heavily influenced by the gladius51 and more than 50% of the characters are based on this feature1,3. Indeed, the authors1 note that the lack of gladius data for some fossil forms, including V. rhodanica, creates an inherent bias in the phylogenetic matrix. Fischer & Riou16 suggested that V. rhodanica and V. infernalis are related on the basis of the observable morphological characters in the family Vampyroteuthidae, though without morphological information on the gladius, a recent systematic synthesis of fossil Octobrachia22 positioned V. rhodanica as Vampyromorpha Incertae sedis.X-ray CT analysis in this study did not allow a reconstruction of the gladius. Nevertheless, it does provide new data on soft tissues, and permits comparisons between extant and fossil taxa. Specifically, we can add distinct states to 4 of the 132 characters in the existing phylogenetic matrix from Sutton et al.3 that was modified and used in Kruta et al.1. These four characters (#89–#92) represent the suckers, and sucker attachments. Detailed examination revealed that the sessile and dorsal arms have the Vampyroteuthis-like attachment. In the dorsal arms, this is more elongated, though it cannot be considered pedunculate like those seen in modern decabrachians. Indeed, the attachment type (plug and base34) is the same, only the length varies. As previously discussed, this variation may have functional implications.When updated with these new data, the matrix from this study returns the same topology seen in Kruta et al.1 that supports the positioning of V. rhodanica and V. infernalis as sister taxa. Further, it strengthens their relationship as they both share a sucker attachment that is not clearly attached to the arm muscles, a state that was previously considered autapomorphic in V. infernalis. However, it is important to note that no additional characters were added for the gladius, which is the cornerstone of coleoid systematics52. Indeed, just 29 of the 132 matrix characters can so far be coded for V. rhodanica, with only 9 of these relating to the 74 states of the gladius.Assuming the phylogenetic work so far is correct, then both species belong to the family Vampyromorphina, and are joined by the Oligocene fossil Necroteuthis hungarica29. While the lack of gladius characters precludes a full phylogenetic understanding of this group, preservation and observation of the soft tissues allow us to infer information regarding palaeobiology.The data obtained in this study demonstrates that the characters observed in V. infernalis, including the sucker attachments and lack of ink sac, were present in Jurassic Vampyromorpha. Comparative anatomy of V. rhodanica and extant V. infernalis revealed that the fossil taxon displayed more morphological variation and were more diversified than previously understood. The assemblage of characters observed in V. rhodanica are consistent with a pelagic predatory lifestyle and corroborate the likelihood of a distinctly different ecological niche. These findings support the hypothesis that a shift towards a deep-sea environment occurred prior to the Oligocene5,29. More

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Study speciesCane toads (Rhinella marina) are large (to  > 1 kg) bufonids (Fig. 1a). Although native to north-eastern South America, these toads have been translocated to many countries worldwide to control insect pests12. Adult cane toads forage at night for insect prey and retreat to moist shelter-sites per day13. Small body size (and thus, high desiccation rate) restricts young toads to the margins of natal ponds14, but adult toads can survive even in highly arid habitats if they have access to water13,15. Cane toads prefer open habitats for foraging12, and thus can thrive in post-fire landscapes16,17. Cane toads in post-fire landscapes tend to have lower parasite burdens, probably because free-living larvae of their lungworm parasites cannot survive either the fire or the more sun-exposed post-fire landscape18.Figure 1taken from study sites between Casino, Grafton, and surrounds, NSW, by S.W. Kaiser.The cane toad Rhinella marina (a), and unburned, (b) and burned (c) habitats in which toads were collected and radio-tracked. Photographs were Full size imageStudy areaEast of the Great Dividing Range, near-coastal Clarence Dry Sclerophyll Forests of north-eastern New South Wales (NSW) are dominated by Spotted gum (Corymbia variegata) and Pink bloodwood (Corymbia intermedia)19. Fires are common, but typically cover relatively small areas before they are extinguished. In the summer of 2019–2020, however, prolonged drought followed by an unusually hot summer resulted in massive fires across this region, burning almost 100,000 km2 of vegetation9. In the current study, the toads we measured and dissected came from several sites within 75 km of the city of Casino (for site locations, see Fig. 2, Table 1, and18). The impacts of fire on faunal abundance and attributes shift with time since fire; for example, the abundance of a particular species may be reduced by fire (due to mortality from flames) but then increase as individuals from surrounding areas migrate to the recently-burned site to exploit new ecological opportunities provided by that landscape8. We chose to study this system 1-year post-fire, to allow time for such longer-term effects to be manifested.Figure 2Sampling sites relative to fire history. Sample sites are burned (red circles), and unburned (green squares). See Table 1 for key to sites. The legend shows the extent of burn a year prior to our study. Map created in QGIS 3.22.3. Fire history available from https://datasets.seed.nsw.gov.au/dataset/fire-extent-and-severity-mapping-fesm CC BY 4.0.Full size imageTable 1 Sampling sites and sample sizes for dissected and radio-tracked cane toads (Rhinella marina) in New South Wales, Australia.Full size tableSurveys of toad abundanceTo quantify toad abundance in burned and unburned sites, one observer (MJG) walked 100-m transects along roads at night (N = 23 and 8 respectively), recording all toads and native frogs (both adult and juvenile). The smaller number of unburned sites reflects the massive spatial scale of the wildfires, which made it difficult to find unburned areas. The transect sites were not the same as those sampled by “toad-busters” (below). We sampled both burned and unburned sites on each night, to de-confound effects of weather conditions with fire treatment. We scored frogs as well as toads to provide an estimate of overall anuran abundance and activity, and so that we could examine toad abundance relative to frog abundance as well as absolute toad numbers.“Toad-buster” sampleBecause of their ecological impact on native fauna, cane toads are culled by community groups as well as by government authorities12,20. We asked “toad-buster” groups to record whether the sites at which they collected toads had been burned during the 2019–2020 fires, or had remained unburned (Table 1). The toads were humanely euthanized (cooled-then-pithed: see21). The euthanasia method is brief (a few hours in the refrigerator, followed by pithing) and thus should not have affected any of the traits that we measured. For all of these toads, we measured body length (snout-urostyle length = SUL) and mass, and determined sex based on external morphology (skin colour and rugosity, nuptial pads: see22). A subset of toads (chosen to provide relatively equal numbers of males and females, and with equal numbers from burned and unburned sites) was dissected to provide data on mass of internal organs (fat bodies, liver, ovaries), reproductive condition (state of ovarian follicle development) and diet (mass and identity of prey items). To select the subsample of toads for dissection, we took relatively equal numbers of male and female toads from each bag of toads that was provided to us by the “toad-busters”. For logistical reasons, we were unable to dissect all of the toads that had been collected. Overall, we obtained data on morphology, diets and other traits from 481 fully dissected and 1443 partially dissected cane toads.Radio-trackingTo explore habitat use and movement patterns, we radio-tracked 57 toads over the course of two fieldtrips (0900–1800 h from 20 Nov 2021 to 6 Dec 2021 and 25 Jan 2022 to 10 Feb 2022). We selected seven sites (4 burned, 3 unburned) within 28 km of Tabbimoble, NSW (see Table 1 for locations and sample sizes of tracked toads). We hand-captured toads found active at night. These were measured, and their sex determined by external morphology (see above) and behaviour (release calls, given only by males: see23). We then fitted the toads with radio-transmitters (PD-2; Holohil Systems, Ontario, Canada; weighing ≤ 3.8 g) on cotton waist-belts, and released them at the site of capture. Tracked toads were 88.2–160.9 mm SUL (mass 70.1–546.3 g); thus, transmitters weighed  20 mm thick) within the quadrat, and estimated exposure of the toad within its refuge (the percentage of the animal’s body exposed to the naked eye). We then selected a compass bearing at random and walked 20 m in that direction where we rescored all of the above habitat attributes, to quantify habitat features in the broader environment (i.e., not just in microhabitats used by toads). We used those “random” sites to quantify overall habitat attributes of burned and unburned sites. Temperature was recorded by directing a temperature gun (Digitech QM7221) on (or otherwise close-to) toads and at a random point on the ground for random replicates. In total, we gathered radio-tracking data on movements and habitat variables from 57 cane toads, each of which was tracked for 5 days. Recaptured toads were euthanized by cooling-then-pithing.Morphological traitsTo obtain an index of body condition of toads, we regressed ln mass against ln SUL, and used the residual scores from that general linear regression as our estimate of body condition. Negative residual scores show an individual that weighs less-than-expected based on its body length. Likewise, we regressed mass of the fat bodies, liver and stomach against body mass to obtain indices of energy stores and stomach-content volumes relative to body mass. We scored male secondary sexual characteristics using the system of Bowcock et al.22. In their system, three sexually dimorphic traits (nuptial pad size, skin roughness and skin colouration) are scored from 0 to 2, and the scores from those three traits are summed to create a final value (on a 6-point scale) for the degree of elaboration of male-specific secondary sexual characteristics. We scored reproductive condition in adult female toads based on whether or not egg masses were visible during dissection, based on dissected toads from both “toad-buster” and telemetry samples.Statistical methodsData were analysed in R version 4.2.025. We used Linear Mixed Models (LMMs), Generalised Linear Mixed Models (GLMMs) and logistic regressions for our analyses. The R packages ‘tidyverse’26, ‘lmerTest’27, and ‘performance’28 were used.Habitat dataWe compared habitat variables between burned and unburned sites, and attributes of toads in burned versus unburned sites, using GLMMs (with negative binomial distribution) for count data (models were checked for overdispersion29) and LMMs on distance data, using ln-transformations where required to achieve normality. LMMs were used on non-normal percentage data, which were ln- and then logit-transformed (using log[(P + e)/(1 − P + e)], where e is the lowest non-zero number, halved)30. We used toad id, site (sampling location) and sampling trip (2019 versus 2020) as random factors.Anuran transect dataCounts of toads in burned versus unburned areas were compared both directly via GLMMs with a negative binomial distribution and relative to the numbers of frogs sighted along the same transects (binding the columns in R as ‘number of toads, number of amphibians – number of toads’ and using a GLMM with a binomial distribution). We used site as a random factor.Telemetry dataFor telemetry data, we analysed response variables via LMMs, and ln-transformed data where relevant to achieve normality.Dissection dataWe used LMMs for SUL, body mass, body condition and organ mass residuals (e.g., fat body mass relative to body mass). For prey item data, we used a poisson distribution with row number as a random factor, as the negative binomial and beta distribution GLMMs were overdispersed (see31). We used LMM for number of prey items and number of prey groups, with site as a random factor. Where models failed to converge, we reduced or removed the error term(s). Analyses were restricted to toads ≥ 70 mm SUL, because animals below this size were difficult to sex. We also performed nominal logistic regression to explore variation in sex ratio and male secondary sexual traits.Reproductive conditionWe used LMM for male secondary sexual characteristic display, using site as a random factor. For ovary presence, we used a binomial GLMM with a logit link, using site as a random factor. We used a LMM of the residual values from ovary mass relative to body mass (ln-transformed), using site as a random factor.Ethics declarationsAll procedures were performed in accordance with the relevant guidelines and regulations approved by Macquarie University Animal Ethics Committee (ARA Number: 2019/040-2) and in accordance with ARRIVE guidelines. More

DEGs under low-temperature stressThe results from the field experiments indicated that the daily mean values of A, Fvʹ/Fmʹ, ETR and Fv/Fm decreased in the LT group, the PSII function was impaired, and the photosynthetic capacity was weakened. Through the specific analysis of the “Photosynthesis” pathway (pathway ID ko00195) in the LT group, it was found that PSII, the cytochrome b6f. complex (Cyt b6f.), PSI and ATPase exhibited differential gene expressions. Figure 9 shows the structural pattern diagram for photosynthesis. The parts marked by white boxes indicate that the structure has DEGs. The gene expressions of CP43, CP47, D1 protein and Cytb559 of PSII changed. The inner peripheral antenna pigment proteins, CP43 and CP47, of PSII bind to chlorophyll. They accept the excitation energy transferred from the surrounding antenna complex and transfer this energy to the reaction centre complex. Changes in CP43 and CP47 affect the absorption and transmission of light energy. In the PSII reaction centre, light energy is converted into chemical energy. P680 absorbs light and is excited to become P680*, and then transfers electrons to pheophytin (Pheo). At the same time, the PSII oxygen-evolving complex obtains electrons from water molecules, the water molecules are split and releases oxygen and protons. As one of the two core proteins that compose the reaction centre complex, the D1 protein combines with various cofactors that are related to the original charge separation and electron transfer. The D1 protein plays an important role in the process of photosynthetic electron transfer. Studies have found that low temperatures can induce allosteric inactivation of the D1 protein, which results in changes in the structure of thylakoid membranes and hinders electron transfer8. As part of the reaction centre, Cytb559 can adjust the photoinhibition sensitivity of PSII through redox changes so that the PSII reaction centre is protected from damage9. The light energy absorption, energy conversion and electron transfer functions of PSII are impaired, which result in significant decreases in Fv/Fm to levels far below the normal value. The results of Xiangchun Song are similar to those presented in this paper: the PS II reaction centre of A. mongolicus seedlings is irreversibly inactivated or the thylakoid membrane is damaged under subzero low temperature stress, which may produce serious photoinhibition. However, Song believes that the peripheral antenna component of the optical system is more affected than the core complex at low temperatures, which was not observed in the corresponding results in this study10.Figure 9Photosynthesis of A. mongolicus under low-temperature stress. The areas outlined by white boxes indicate the differentially expressed genes in these structures.Full size imageThe gene expressions of Cyt b6, PrtD and Cyt f in Cyt b6f. changed. Cyt b6f. changes not only affect the electron transport function of photosynthesis but also affect ATP synthesis. Pheo transfers the received electrons to plastid quinone (PQ). PQ receives electrons and protons to form plastid hydroquinone (PQH2). Then, the electrons of PQH2 are transferred to plastid cyanin (PC) on PSI through Cyt b6f., and hydrogen protons are released into the cavity of the thylakoid to form a transmembrane proton gradient. The transmembrane proton gradient is the driving force for ATP synthesis.The function of PSI is to transfer electrons from PC to ferredoxin for the reduction of NADP+. Recent studies have found that PSI is more sensitive to light and more prone to selective photoinhibition than PS II under low temperature and weak light conditions11,12. The KEGG analysis results indicated that the LHCI complex, PsaF and PsaE subunits of PSI showed differential gene expressions. The main function of the LHCI light-harvesting pigment protein complex is to capture light energy. PsaF is a low-molecular-weight protein that is distributed in the membrane. Some studies have suggested that the N-terminal amino acid sequence of eukaryotic PsaF is involved in the binding of PSI and PC13. PsaE, PsaD and PsaC together form the docking site of ferredoxin on the PSI receptor side14,15. Ferredoxin and ferredoxin-NADP+ reductase in the photosynthetic electron transport chain are also affected, which results in hindrance of NADPH synthesis. The F-type H+/Na+ transport ATPase subunits also show differential gene expressions, which lead to impaired ATP synthesis. Low temperatures affect the ability to absorb light energy, transfer electrons, convert light energy into electric energy, and synthesize NADPH as well as ATP, which ultimately lead to declines in Fv’/Fm’ and ETR and impair the photosynthesis capacity of A. mongolicus.Compared with the light reaction, low temperatures have a greater impact on the dark reaction. Because the dark reaction process is composed of many complex enzymatic reactions, the enzyme activity is very susceptible to temperature. The KEGG results show that 13 related enzymes were differentially expressed in the “carbon sequestration of photosynthesis” (ko00710). The Rubisco enzyme is a key enzyme that determines the direction and efficiency of photosynthetic carbon metabolism in C3 plants and is sensitive to temperature16. The results also show that the expression levels of 10 differentially expressed genes of Rubisco enzymes all declined. In the Calvin cycle, the gene expressions of only transketolase and glyceraldehyde-3-phosphate dehydrogenase are not sensitive to temperature. In addition, the reduction phase of the dark reaction requires the use of NADPH and ATP that are produced by the light reaction. The inhibition of NADPH and ATP synthesis will inevitably affect the normal progression of the Calvin cycle.Chloroplast respiration is an O2-dependent electron transport pathway in chloroplasts. Chloroplast respiration includes the nonphotochemical reduction of PQ by NAD(P) H and the reoxidation of PQ by terminal oxidase, which can consume excess electrons to protect plants from damage due to photooxidation.Figure 10 shows the partial KEGG enrichment metabolic pathway in the LT group. There were three significant enrichment pathways related to carbohydrate metabolism: fructose and mannose metabolism (ko00051), butanoate metabolism (ko00650) and C5-branched dibasic acid metabolism (ko00660). The metabolism of fructose and mannose includes the ascorbic acid biosynthetic pathway. Ascorbic acid (ASA), also known as vitamin C, can be used as a cofactor of violaxanthin de-epoxidase to participate in the lutein cycle and consume excess light energy and protect plants from harm.Figure 10The regulatory mechanism of A. mongolicus under low-temperature stress. The white ovals represent the enriched metabolic pathways. The blue rectangles represent significantly enriched KEGG metabolic pathways. The pathways are followed by the physiological structures and substances or physiological processes in which the expressions of related genes change.Full size imageLow temperatures damage cell membranes first. Increasing the mass fraction of unsaturated fatty acids in the membrane is beneficial to improve the stability and fluidity of the membrane. Some studies have shown that the degree of unsaturation of fatty acids in adult leaves of A. mongolicus that grow naturally in the field is lower in summer and higher in autumn and winter17. The significantly enriched pathways related to unsaturated fatty acid metabolism were alpha-linolenic acid metabolism (ko00592), linoleic acid metabolism (ko00591) and arachidonic acid metabolism (ko00590). Various proteins, such as linoleate 13S-lipoxygenase and cytochrome P450 family 2 subfamily J (CYP2J), which are involved in the metabolism of linoleic acid, showed differences in their gene expressions. Linoleate 13S-lipoxygenase is a common lipoxygenase in plants that can catalyse the production of precursors of several important compounds, including jasmonic acid. CYP2J is a group of P450 haem thiolate proteins, which are mainly distributed on the endoplasmic reticulum and inner mitochondrial membrane and are involved in the synthesis of sterol hormones, including brassinosteroids. Because light systems are distributed on the thylakoid membrane, damage to this membrane will affect the progress of plant photosynthesis.Plant hormone signal transduction (ko04075) plays an important role in plant resistance to stress. Studies have shown that JAs have physiological functions, such as inducing stomatal closure, inhibiting photosynthesis, promoting respiration and promoting leaf senescence18,19. Treating plants with exogenous methyl jasmonate can induce the transcription of the heat shock protein family, increase the synthesis of antioxidants, reduce lipoxygenase activity and enhance the ability of plants to resist cold damage20.Figure 11 shows the regulatory mechanism of A. mongolicus in the HL group. The MapMan analysis results show that the DEGs of the LHCII complex and those for the assembly and maintenance of PSII are significantly changed. LHCII contains chlorophyll and carotenoids, which can capture and transmit light energy. Chlorophyll is an important photosynthetic pigment that captures light energy and drives electrons to the reaction centre. The chlorophyll molecule in the reaction centre is related to photochemical quenching. The entire chlorophyll biosynthesis process (e.g., L-glutamyl-tRNA → chlorophyll a → chlorophyll b) involves 15 enzymes. The analysis found that 4/5 of the enzymes’ expression genes were changed. Carotenoids include carotene and lutein, and their synthesis is affected by high temperatures. Lutein participates in the lutein cycle, which can dissipate excess light energy and prevent membrane lipids from being peroxidized and thus maintain the stability of the thylakoid membrane structure and protect A. mongolicus. from high temperature stress and strong light stress.Figure 11The regulatory mechanism of A. mongolicus. under high-temperature stress. The white ovals represent enriched metabolic pathways. The red rectangles represent significantly enriched KEGG metabolic pathways. The pathways are followed by the physiological structures and substances or physiological processes in which the expressions of related genes change.Full size imageThe D1 protein in the PSII reaction centre is rapidly degraded under strong light conditions. To maintain the normal physiological needs of plants, the degraded D1 protein will be replaced by the new D1 protein that is produced by the repair mechanism. The reversible inactivation of the PSII reaction centre can protect the photosynthetic system and avoid destruction. This may be the reason for the significant changes in the DEGs that are involved in the assembly and maintenance of PSII.Rubisco is the main site for high-temperature inhibition of the Calvin cycle16. The KEGG analysis found that there were 7 (4↑, 3↓) DEGs of Rubisco. SBPase catalyses the conversion of sedum heptulose-1,7-diphosphate (SBP) into sedum heptulose-7-phosphate (S7P) in the renewal phase. Under low-temperature stress, only transketolase and glyceraldehyde-3-phosphate dehydrogenase remained unchanged in the Calvin cycle. In addition, NDH-mediated cyclic electron transfer may decreased the photooxidation damage that is caused by high-temperature stress by shunting the excess electrons that were generated by the inhibition of CO2 assimilation to the chloroplast respiratory pathway21.In the HT group, the net photosynthetic rates of the leaves showed two peaks on the diurnal change curves, and there was an obvious phenomenon of midday photosynthesis depression. The daily average A values were greater than those of the CK group. These results show that A. mongolicus has a complete photosynthetic structure protection mechanism and can adapt to high-temperature environments. The pathway of significant enrichment related to carbohydrate metabolism in the HT group was the same as that in the LT group. The enrichment degrees of the fructose and mannose metabolic pathways were higher only in the HT group, and C5-branched dibasic acid metabolism and butanoate metabolism were higher in the LT group.Under high temperature and strong light conditions, the balance between production and removal of reactive oxygen species (ROS) in plant cells was broken, and large amounts of reactive oxygen species accumulated in the cells. Active oxygen can cause lipid peroxidation of the biomembrane, enlarge membrane pores, increase the permeability, and affect the spatial structures of enzymes on the membrane, which thus leads to chloroplast destruction. In severe cases, ROS will cause serious injury or even death to plants22. The gene expressions of FabH and acetyl-CoA carboxylase (ACCase) changed during the synthesis of unsaturated fatty acids in the HT group.There are two types of active oxygen scavenging mechanisms in plants. (1) The enzymatic detoxification system: superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). (2) Nonenzymatic antioxidants: ASA, carotenoids, glutathione, mannitol, and flavonoids23.Secondary metabolites result from long-term adaptation of plants to their environments. They can improve the ability of plants to protect themselves, compete for survival, and coordinate the relationship between plants and the environment. The significant enrichment pathways related to the biosynthesis of secondary metabolites in the HT group consisted of phenylpropane biosynthesis (ko00940), flavonoid biosynthesis (ko00941) and isoflavone biosynthesis (ko00943). The phenylpropanoid biosynthesis pathway is one of the three main secondary metabolic pathways in plants. It starts from phenylalanine and generates different phenylpropane metabolites through multistep reactions, such as flavonoids, isoflavones, anthocyanins and lignin24,25. Anthocyanins can protect plants from light damage by quenching free oxygen radicals and reducing the absorption of light energy. Hughes studied 10 species of evergreen broad-leaved trees and found that red leaves containing anthocyanins always maintained higher Fv/Fm levels than green leaves. Fv’/Fm’ is related to nonphotochemical quenching. This means that trees with red leaves rely more on the light-damage defence function of anthocyanins than on the light-damage defence mediated by lutein26.Riboflavin metabolism (ko00740) and biotin metabolism (ko00780) are two significantly enriched cofactors and vitamin metabolic pathways. Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). As a prosthetic group of flavinases, FAD participates in multiple biochemical processes, such as mitochondrial electron transport, photosynthesis, fatty acid oxidation and folate metabolism, in plants27. Riboflavin can induce antioxidant accumulations in plant cells and can also promote plant growth by affecting the ethylene signalling pathway28. Biotin (e.g., VH or VB7), as an essential cofactor for biotin-dependent carboxylase, plays an important role in the life activities of plants. Common biotin-dependent carboxylase enzymes are pyruvate carboxylase (PC) and ACCase. PC is present in the mitochondria and participates in the replenishment mechanism of the tricarboxylic acid cycle. ACCase plays a pivotal role in the feedback regulation of fatty acid synthesis and is the site of action for the feedback regulation of fatty acid synthesis29.The four pathways related to amino acid metabolism showed differences in the HT group. The enrichment degrees of each pathway were as follows: valine, leucine and isoleucine biosynthesis (ko00290)  > biosynthesis of amino acids (ko01230)  > lysine biosynthesis (ko00300)  > glycine, serine and threonine metabolism (ko00260). The branched chain amino acids, valine, leucine and isoleucine and their derivatives, are beneficial to plant growth and plant responses to stress30. As an essential amino acid, lysine metabolism affects many physiological reactions, such as the tricarboxylic acid cycle, abiotic and biotic stress responses, and starch metabolism31. The glycine, serine and threonine metabolic pathways combined with the GO enrichment results showed that the genes related to glycine catabolism and glycine dehydrogenation/decarboxylase activity changed greatly. It is known that when the activity of mitochondrial glycine decarboxylase increases, both photorespiration and photosynthesis will increase32.In terms of hormones, salicylic acid, cytokinin, and abscisic acid (ABA) can improve plant active oxygen scavenging ability. Salicylic acid can decrease the damage to seedlings due to high temperatures by improving the ability of plants to resist oxidative stress and increasing the contents of osmotic adjustment substances in cells33. Salicylic acid also has the function of delaying the degradation of D1 protein and speeding up the recovery of D1 protein when high temperatures are no longer present34. ABA can improve the heat tolerance of plants by regulating the expressions of heat stress-induced genes at the transcriptional level35.In conclusion, A. mongolicus has weak resistance to low temperatures and good adaptation to high temperatures. At the physiological level, under low-temperature stress, the proportion of Y (NO) increased, the function of PSII was damaged, and photosynthesis was inhibited. A. mongolica maintains normal physiological activities by regulating the circadian rhythm, increasing the synthesis of unsaturated fatty acids and changing the effects of plant hormones. Under high-temperature stress, A. mongolicus maintains normal photosynthesis by adjusting gsw as well as water utilization and by increasing the proportion of Y (NPQ). At the same time, A. mongolicus uses LHCII to consume excess energy, continuously assembles and maintains the normal function of PSII, and changes the types of antioxidants, such as by synthesizing anthocyanins, flavonoids, and isoflavones, to protect itself from injury. In addition, the porphyrin and chlorophyll metabolisms, carotenoid metabolism, plant hormones, amino acid metabolism, unsaturated fatty acid synthesis and other metabolic pathways that are related to the differentially expressed genes changed greatly. More