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    Comparative genomic analyses of four novel Ramlibacter species and the cellulose-degrading properties of Ramlibacter cellulosilyticus sp. nov.

    Chemotaxonomic characteristicsThe predominant respiratory quinone for all novel strains was ubiquinone 8 (Q-8), consistent with other Ramlibacter species. C16:0 and summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c) were identified as the common major fatty acids ( > 10%) of the novel strains USB13T, AW1T, GTP1T, and HM2T. Other than the aforementioned fatty acids, strain USB13T had C10:0 3-OH additionally as its major fatty acid, whereas strains AW1T and HM2T shared C17:0 cyclo and summed feature 8 (consisting of C18:1 ω7c and/or C18: 1 ω6c) as its additional fatty acids. Detailed comparisons of the fatty acid profiles of the novel strains and their reference strains are summarized in Table S1.Strains USB13T, AW1T, GTP1T, and HM2T shared major polar lipids diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), which was consistent with the major polar lipids of the reference strains. Additionally, the polar lipid profile of USB13T consisted of one unidentified phosphoaminolipid, two unidentified phosphoglycoaminolipids, and six unidentified polar lipids while the polar lipid profile of AW1T had one unidentified lipid, one unidentified phosphoglycolipid, and three unidentified glycolipids in addition. The polar lipid profile of strain GTP1T additionally consisted of two unidentified phosphoaminolipids, and that of strain HM2T additionally had one unidentified phosphoaminolipid, one unidentified phosphoglycolipid, one unidentified phosphoglycoaminolipid, and two unidentified phospholipids. Polar lipid profiles of the novel strains USB13T, AW1T, GTP1T, and HM2T are shown in Figure S1.Physiological, morphological characteristics, and screening of cellulose-degrading strainsWhen grown on R2A agar, strain USB13T produced reddish white and flat colonies while strain AW1T produced orange, convex colonies, strain GTP1T produced white, convex colonies, and strain HM2T produced cream-colored, flat, transparent colonies. Under TEM, monotrichous flagella were observed only in strain HM2T, and when tested for motility, strain USB13T and AW1T showed gliding motility, whereas strain GTP1T was non-motile. Strains USB13T and HM2T showed positive results for both catalase and oxidase activities; strain AW1T showed positive results for catalase and negative results for oxidase activity, and strain GTP1T showed negative results for catalase and positive results for oxidase activity. All strains were identified to be strictly aerobic, while showing negative results for urea, gelatin, starch, chitin, and DNA hydrolysis and positive results for hydrolysis of Tween 80. In addition, strain USB13T was the only strain to produce iron-chelating siderophores. When tested for NaCl tolerance, growth of strain USB13T was observed in NaCl concentrations of 0–7% (w/v), possibly due to the fact the strain was isolated from a marine environment. A detailed comparison of physiological and morphological characteristics between the novel species and its closely related Ramlibacter strains is presented in Table 1, while TEM images of the novel strains are shown in Figure S2. Results of the reference strains in Table 1 coincided with the data from the original literature1,3,4,5,7,8.Table 1 Characteristics differentiating strains USB13T, AW1T, GTP1T, and HM2T from closely related strains of the genus Ramlibacter.Full size tableStrains: 1, USB13T; 2, AW1T; 3, GTP1T; 4, HM2T; R. monticola KACC 19175T; 6, R. alkalitolerans KACC 19305T; 7, R. ginsenosidimutans KACC 17527T; 8, R. humi KCTC 52922T; 9, R. henchirensis KACC 11925T; 10, R. tataouinensis KACC 11924T; 11, R. rhizophilus KCTC 52083T. All strains are positive for esterase lipase (C8), while all strains are negative for chitin hydrolysis. All data were obtained from this study unless indicated otherwise. + , Positive; w + , weakly positive; -, negative.R2A agar plates supplemented with 1% (w/v) CMC were stained with Congo red dye after 7 days of incubation. Clear zones only formed around colonies of strain USB13T, indicating that strain USB13T solely possessed CMC-hydrolyzing activity among the four novel strains. When inoculated in basal salt medium, filter paper from the USB13T sample underwent degradation, whereas samples containing strains AW1T, GTP1T, and HM2T did not show any signs of degradation.Phylogenetic and genomic analysesEzBioCloud search results and BLASTn searches revealed that the novel strains belonged to the family Comamonadaceae and genus Ramlibacter. Using BLASTn, 16S rRNA gene sequence similarities were determined where strain USB13T was closest to strain GTP1T (98.5%), followed by strain HM2T (98.1%) and strain AW1T (97.1%). Strain AW1T shared the highest similarity with strain GTP1T (97.3%), followed by strain HM2T (97.1%), while strain GTP1T shared a similarity of 98.2% with strain HM2T. Phylogenetic analysis based on the MP method (Fig. 1) showed the clustering of the novel strains USB13T, AW1T, GTP1T, and HM2T with strains such as R. monticola G-3-2T, R. ginsenosidimutans BXN5-27T, R. alkalitolerans CJ661T, and R. rhizophilus YS3.2.7T. Similar topologies were observed in trees reconstructed by ML (Figure S3) and MP methods. The UBCG phylogenomic tree (Fig. 2), which was reconstructed using whole genome sequences, also showed close clustering of the selected reference strains and novel strains.Figure 1Maximum-parsimony (MP) tree reconstructed based on 16S rRNA gene sequences, showing the relationship between strains USB13T, AW1T, GTP1T, and HM2T and other closely related type strains. Bootstrap values based on 1000 replications are listed as percentages at branching points. Only bootstrap values exceeding 50% are shown. Bar, 50 substitutions per nucleotide position.Full size imageFigure 2Phylogenomic tree of strains USB13T, AW1T, GTP1T, and HM2T and their closely related taxa was reconstructed based on core genomes using UBCG version 3.0 pipeline42. NCBI GenBank accession numbers are shown in parentheses. Bootstrap analysis was carried out using 1000 replications. Percentage bootstrap values ( > 50%) are given at branching points. Bar, 0.050 substitution per position.Full size imageDraft genome sequences of the novel strains USB13T, AW1T, GTP1T, and HM2T were deposited in the GenBank database under the accession numbers JACORT000000000, JAEQNA000000000, JACORU000000000, and JADDIV000000000, respectively. In addition, the draft genome sequences of R. monticola KACC 19175T, R. alkalitolerans KACC 19305T, and R. ginsenosidimutans KACC 17527T were also deposited in GenBenk under the accession numbers JAEQNE000000000, JAEQND000000000, and JAEPWM000000000, respectively. The assembled genome size of the novel strains USB13T, AW1T, GTP1T, and HM2T was 5.53 Mbp, 5.11 Mbp, 6.15 Mbp, 4.31 Mbp, respectively. G + C content ranged from 67.9% to 69.9%, which was similar to those of the reference strains. The genomic features of the novel strains and their closely related Ramlibacter strains are presented in Table S2. CheckM analysis showed the following estimations for each strain: USB13T, had a 99.84% completeness and 0.68% contamination; AWIT, had a 99.84% completeness and 0.86% contamination; GTP1T, had a 99.38% completeness and 1.32% contamination; HM2T, had a 97.51% completeness and 0.16% contamination. These results indicated that the draft genome results for all strains were reliable. ANI values between the novel strains and reference strains ranged from 76.5–83.4% while dDDH values ranged from 20.7–26.7%, and AAI values ranged from 65.7–80.4%. All values were below the threshold for delineation of a new species54. ANI values between the novel strains and their reference strains are presented in Fig. 3, while a detailed comparison of GGDC and AAI values are shown in Table 2.Figure 3Heatmap of strains USB13T, AW1T, GTP1T, and HM2T and other closely related strains within the genus Ramlibacter, generated with OrthoANI values calculated using OAT software45. Bacterial strains and accession numbers are indentical to those of Fig. 2.Full size imageTable 2 Average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) value comparisons between the closely related Ramlibacter type species and the novel strains, USB13T, AW1T, GTP1T, and HM2T. AAI values were calculated by two-way AAI, while dDDH values were calculated based on formula 246.Full size tableBased on NCBI PGAP annotation and CAZyme prediction results, strain USB13T, which was the only strain to show cellulolytic activity, possessed a total of four protein CDs encoding CAZymes, namely, two GH15 proteins, one glycosyl hydrolase protein, and one GH99-like domain-containing protein. Despite not showing any cellulolytic activity, strain AW1T possessed eight CAZyme CDs; the most amount among the novel strains. The enzymes include, two GH2 proteins, one GH5 protein, three GH15 proteins, one glycoside hydrolase protein, and one cellulase family glycosyl hydrolase. Strain GTP1T possessed two CDs encoding one GH15 protein and one GH16 protein; strain HM2T possessed three CDs encoding one GH2, one GH15, and one GH18 protein. All strains possessed GH15, which is known for its glucoamylase activity in fungi55. A detailed summary of the novel strains CAZymes are presented in Table S3 and a comparison of CAZyme numbers between strains USB13T, AW1T, GTP1T, and HM2T is summarized in Table S4. The presence of these genes may suggest the cellulolytic activity of strain USB13T, while it is uncertain why GH families responsible for endoglucanase (GH 5–8, 12, 16, 44, 45, 48, 51, 64, 71, 74, 81, 87, 124, and 128), exoglucanase (GH 5–7, and 48), and β-glucosidase (GH 1, 3, 4, 17, 30, and 116) were not present in the genome11.COG predictions (Fig. 4) revealed that the majority of the core genes of the four novel strains accounted for genes belonging to the functional categories C (energy production and conversion), E (amino acid transport and metabolism), I (lipid transport and metabolism), T (signal transduction mechanisms), and K (transcription). Meanwhile, the number of core genes belonging in category G, carbohydrate transport and metabolism, was the highest for strain USB13T (258), followed by GTP1T (230), HM2T (212), and AW1T (181). The high number of genes in strain USB13T may be a contributing factor in the strain’s cellulolytic activity. A comparison of COG gene count distribution of the novel strains is presented in Table S5.Figure 4Comparison of total number of matched genes of strains USB13T, AW1T, GTP1T, and HM2T according to functional classes based on Cluster of Orthologous Groups of proteins (COG) predictions48.Full size imageAntiSMASH analysis results showed four gene clusters within the genome of strain USB13T: ribosomally synthesized and post-translationally modified peptides (RIPP)-like cluster (989,516–1,000,916 nt; JACORT010000001), terpene synthesis (8,622–30,347 nt; JACORT010000003), RIPP precursor peptide recognition element (RRE)-containing cluster (311,469–333,619 nt; JACORT010000004), and redox-cofactor (281,860–303,948 nt; JACORT010000007). Among the clusters, the RRE-containing cluster showed 11% similarity to streptobactin, a tricatechol-type siderophore isolated from Streptomyces sp. YM5-79956. Strain AW1T had a total of eight gene clusters which encoded for: arylpolyene (165,946–207,130 nt), terpene (618,322–640,854 nt), RIPP-like proteins (804,411–819,137 nt), non-ribosomal peptide synthetase cluster (NRPS)-like (61,798–104,764 nt), betalactone (323,399–348,739 nt), N-acetylglutaminylglutamine amide (NAGGN; 106,834–121,648 nt), type I polyketide synthase (T1PKS; 56,584–107,578 nt), and heterocyst glycolipid synthase-like polyketide synthase (hglE-KS; 75,419–113,566 nt). Strain GTP1T possessed four gene clusters that encoded for RRE-containing cluster (175,155–199,102 nt), homoserine lactone (110,293–130,892 nt), a signaling molecule known for its involvement in bacterial quorum sensing, the RIPP-like cluster (38,002–48,856 nt), and terpene synthesis (47,942–69,701 nt). Strain HM2T had two gene clusters that encoded for resorcinol (403,967–445,901 nt), an organic compound known for its antiseptic properties, and terpene (697,660–721,242 nt), which showed 100% similarity for carotenoid synthesis. BRIG analysis results showed that a majority of the regions within the four analyzed genomes were conserved with at least 70% similarity (Figure S4).Cellulolytic potential and FE-SEM analysis of strain USB13T
    A USB13T-inoculated basal salt medium sample containing degraded filter paper was examined under FE-SEM to observe the morphological interactions between cellulose fibers and USB13T cells. Images in Fig. 5 show individual rod cells of strain USB13T surrounding filter paper fibers, indicating bacterial adherence.Figure 5Field emission-scanning electron microscopy (FE-SEM) images of adhesion of strain USB13T to degraded filter paper fibers. Arrows indicate filter paper fibers. (A) low magnification (5000(times)) and (B), high magnification (20,000(times)) images of strain USB13T surrounding filter paper fibers.Full size imageThe enzymatic assay results showed endoglucanase, exoglucanase, β-glucosidase, and filter paper cellulase (FPCase) activities of strain USB13T, wherein activities for endoglucanase was the highest and β-glucosidase was the lowest in all experiments. As seen in Fig. 6A, enzyme activity for all cellulolytic enzymes increased along with its cultivation time. In addition, enzyme activities showed the highest results when tested on buffer solutions of pH 6.0 (Fig. 6B), indicating the enzymes’ resistance to moderately acidic conditions. The pH of the buffer solution seemed to be an important factor in enzyme activity, as activity of endoglucanase, exoglucanase, and FPCase drastically decreased when the pH was altered from pH 6.0 to pH 7.0. Meanwhile, β-glucosidase activity was relatively resistant to pH change as its activity decreased less than 50%. On day 7, enzyme activities were measured as 1.91 IU/mL for endoglucanase, 1.77 IU/mL for exoglucanase, 0.76 IU/mL for β-glucosidase, and 1.12 IU/mL for FPCase at pH 6.0. When measured at pH 8.0, where enzyme activity was the lowest, enzyme activities were measured as 0.51 IU/mL for endoglucanase, 0.25 IU/mL for exoglucanase, 0.45 IU/mL for β-glucosidase, and 0.23 IU/mL for FPCase; all values were less than half of the measured activity at pH 6.0. The results of strain USB13T are comparable to FPCase results of other species such as Mucilaginibacter polytrichastri RG4-7T (0.98 U/mL) isolated from the moss Polytrichastrum formosum14, Paenibacillus lautus BHU3 (2.9 U/mL) isolated from a landfill site57, and Serratia rubidaea DBT4 (0.5 U/mL) isolated from the gastrointestinal tract of a black Bengal goat58.Figure 6Cellulolytic enzyme activity of strain USB13T. Enzyme activity was defined in international units (IU); one unit of enzymatic activity was defined as the amount of enzyme that releases 1 μmol of glucose per mL per 1 min of reaction. (A) cellulase activity results under different cultivation time; (B) cellulase activity under different buffer solution pH. Values in the figure are mean values of triplicate data with standard deviation.Full size imageDespite the absence of the main three cellulolytic enzymes, endoglucanase, exoglucanase, and β-glucosidase, the cellulolytic activity of strain USB13T was confirmed through SEM images, CMC agar screening, and enzymatic assay results. However, because PGAP annotation results showed that other non-cellulolytic strains also possessed CAZymes, in some cases more than strain USB13T, further research is necessary to understand the mechanics of how CAZymes and other cellulases interact to degrade cellulose, and how these genes are expressed under certain conditions. Furthermore, the cellulolytic activity of strain USB13T can be further optimized for commercial use by adjusting growth conditions such as pH, temperature, and growth media.While cellulolytic bacteria are known to inhabit animal intestinal tracts, the rumen, and soil, they can be found almost everywhere, such as ocean floors, municipal landfills, and even extreme environments such as hot springs59. In these habitats, cellulolytic bacteria utilize cellulose while cohabiting with non-cellulolytic bacteria. There have been many studies suggesting the synergistic role non-cellulolytic bacteria play in cellulose degradation, where non-cellulolytic bacteria aid cellulose degradation by neutralizing pH or removing harmful metabolites60,61,62.Bacterial cellulases have shown immense value in various industries such as animal feed processing, food and brewery production, and agriculture, not to mention biofuel synthesis through biomass utilization11. Due to the versatile uses of bacterial cellulases, the cellulolytic strain USB13T has the potential to become an invaluable resource. However, further research of the novel strain’s cellulose-degradation mechanisms is necessary to develop and commercially make use of its bacterial cellulases in the future. In addition, research regarding co-culturing non-cellulolytic bacteria and strain USB13T may also help in developing effective methods to use an otherwise underutilized bioresource.Taxonomy of novel Ramlibacter speciesWhile phylogenetic analyses indicated that the novel strains USB13T, AW1T, GTP1T, and HM2T should be assigned to the genus Ramlibacter, differences in fatty acid compositions, polar lipid profiles, and physiological characteristics suggested that the four novel strains are noticeably distinct from other validly published species of the genus. Additionally, genomic characteristics such as ANI, dDDH, and AAI values further supported the novel strains’ position as a distinct species within the genus Ramlibacter. Therefore, we propose that the strains USB13T, AW1T, GTP1T, and HM2T represent novel species within the genus Ramlibacter.Description of the novel Ramlibacter speciesThe descriptions of the novel species are given according to the standards of the Judicial Commission of the International Committee on Systematic Bacteriology63.Description of Ramlibacter cellulosilyticus sp. nov
    Ramlibacter cellulosilyticus (cel.lu.lo.si.ly’ti.cus. N.L. n. cellulosum, cellulose; N.L. adj. lyticus from Gr. lytikos, dissolving; N.L. masc. adj. cellulosilyticus, cellulose-dissolving).Cells of strain USB13T are Gram-negative, rod-shaped, non-flagellated and motile by gliding. The strain is positive for both oxidase and catalase activity, while cells have a width of 0.3–0.5 μm and length of 2.0–2.4 μm. When observed on R2A agar, colonies are reddish white, flat with entire margins, and have a diameter of 1–2 mm. Growth of strain USB13T is observed at 7–50 °C (optimum, 28–30 °C), at pH 5.0–10.0 (optimum, pH 6.0), and at NaCl concentrations of 0–7% (optimum, 0–3%). The strain is unable to grow in anaerobic conditions. Produces siderophores and hydrolyzes Tween 20, Tween 80, CMC, and esculin. According to the API ZYM results, the strain showed positive results for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, acid phosphatase, β-galactosidase, α-glucosidase, and β-glucosidase. In the API 20NE assay, strain USB13T showed positive results only for β-galactosidase. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C16:0, C10:0 3-OH, and summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoaminolipid, two unidentified phosphoglycoaminolipids, and six unidentified polar lipids. The G + C content is 69.7%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain USB13T are MN603953 and JACORT000000000, respectively.The type strain USB13T (= KACC 21656T = NBRC 114839T) was isolated from shallow coastal water at Haeundae Beach, Busan, Republic of Korea.Description of Ramlibacter aurantiacus sp. nov
    Ramlibacter aurantiacus (au.ran.ti’a.cus. L. masc. adj. aurantiacus, orange-colored, referring to the orange colonies of the strain).Cells of strain AW1T are Gram-negative, coccoid to short rod-shaped, non-flagellated, and motile by gliding. The strain is negative for oxidase activity, and positive for catalase activity. When observed on R2A agar, colonies are orange, convex, with entire margins, and 0.5–1.0 mm in diameter. Under TEM cells have and approximate width of 0.3–0.5 μm and length of 0.6–0.8 μm. Growth of strain AW1T can be observed at 7–45 °C (optimum, 30 °C), at pH 7.0–10.0 (optimum, 7.0–8.0), and at NaCl concentrations of 0–3% (optimum, 0–1%). The strain does not grow under anaerobic conditions but is able to hydrolyze Tween 80. In addition, AW1T is not able to produce siderophores. In the API ZYM assay, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, and β-glucosidase. In the API 20NE assay, positive for esculin hydrolysis. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C16:0, C17:0 cyclo, summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c), and summed feature 8 (consisting of C18:1 ω7c and/or C18:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoglycolipid, one unidentified lipid, and three unidentified glycolipids. The G + C content is 68.6%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain AW1T are MN498045 and JAEQNA000000000, respectively.The type strain AW1T (= KACC 21544T = NBRC 114862T) was isolated from soil at Aewol, Jeju Island, Republic of Korea.Description of Ramlibacter albus sp. nov
    Ramlibacter albus (al’bus. L. masc. adj. albus, white, referring to the white colonies of the strain).Strain GTP1T is non-motile, Gram-negative, strictly aerobic, positive for oxidase activity, and negative for catalase activity. When observed on R2A, colonies are white, convex, with entire margins, and 1–2 mm in diameter. Under TEM, cells lack flagella, are rod-shaped, and have a width of 0.7–0.8 μm and length of 1.6–1.9 μm. Growth of strain GTP1T can be observed at 10–45 °C (optimum, 30 °C), at pH 5.0–8.0 (optimum, pH 7.0), and at NaCl concentrations of 0–2% (optimum, 0%). The strain shows positive results for Tween 20 and Tween 80 hydrolysis. GTP1T does not produce siderophores when tested on CAS-blue agar. According to API ZYM results, strain GTP1T is positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), and leucine arylamidase, while the API 20NE assay results show negative results for all substrates. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C16:0 and summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and two unidentified phosphoaminolipids. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C16:0, C17:0 cyclo, summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c), and summed feature 8 (consisting of C18:1 ω7c and/or C18:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoaminolipid, one unidentified phosphoglycolipid, one unidentified phosphoglycoaminolipid, and two unidentified polar lipids. The G + C content is 67.9%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain GTP1T are MN498046 and JACORU000000000, respectively.The type strain GTP1T (= KACC 21702T = NBRC 114488T) was isolated from soil at Seogwipo, Jeju Island, Republic of Korea.Description of Ramlibacter pallidus sp. nov
    Ramlibacter pallidus (pal’li.dus. L. masc. adj. pallidus, pale, referring to the color of the colonies).Cells of strain HM2T are Gram-negative, and positive for both oxidase and catalase activities. When observed on R2A agar, colonies are cream-colored, transparent, 1.0–2.5 mm in diameter, and flat with entire margins. Under TEM, monotrichous flagella are observed, and cells are rod-shaped with a width of 0.4–0.78 μm and length of 1.7–1.8 μm. The strain shows the fastest growth at a temperature range of 25–35 °C and at pH values between 8.0 and 9.0. When NaCl is present, growth is observed at concentrations of 0–3% (w/v), with optimal growth was observed at concentrations of 0–1% (w/v). The strain is not able to tolerate anaerobic conditions. Strain HM2T hydrolyzes Tween 80 and weakly hydrolyzes casein. However, siderophore production cannot be observed when tested on CAS-blue agar. According to API ZYM tests, strain HM2T shows positive results for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. In addition, API 20NE tests show positive results for nitrate (NO3) to nitrite (NO2-) reduction and esculin hydrolysis. The G + C content is 69.9%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain HM2T are MN498047 and JADDIV000000000, respectively.The type strain HM2T (= KCTC 82557T = NBRC 114489T) was isolated from soil at Seopjikoji, Jeju Island, Republic of Korea. More

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    Biodiversity and climate COPs

    Restoring the connection between people and the rest of nature hinges on whole-system science, actions and negotiations.
    Those who think about and practise sustainability are constantly looking for holistic interpretations of the world and are trying to understand systemic relations, networks and connections. Biodiversity has all of these things. It shows how every species needs other species to exist and thrive. It shows that all living organisms are part of a sophisticated and fascinating system made up of myriads of links. And humans are undoubtedly a part of it.
    Credit: Pulsar Imagens / Alamy Stock PhotoIn the realm of sustainability, experts also ponder about time: how can life exist and thrive over time? Indeed, the above mentioned fascinating system evolves over time. And, over time, it has to adapt to unexpected change. It does that well when it is healthy, and less well when it is ill and constantly disturbed.For a long time, man-made impacts kept accumulating almost completely unchecked by societies, until the consequences for human well-being became untenable. Nowadays, environmental crises make the headlines regularly. They are nothing but the result of a broken connection between people and the rest of nature.Climate change is one major outcome of the broken human–rest of nature connection and has wide ramifications for both people and the planet. We now face imminent disaster, unequally across the world, yet addressing climate change remains an incredibly thorny task. Country representatives from most nations around the world meet regularly at the Conference of the Parties (COP) to the United Nations Framework Convention on Climate Change (UNFCC) — most recently at COP27, which was held in Egypt — to continue the debate on what actions are needed to move the climate agenda forward, all while disasters continue to hit the most vulnerable populations. The world has seen 27 COP meetings to the UNFCC so far; one wonders how many more meetings will be needed to see real change happen.Interestingly, country representatives also meet regularly to discuss biodiversity protection; biodiversity decline — the other major consequence of the broken human–rest of nature connection — is just as worrying, with severe and ramified implications that are still largely underappreciated by decision-makers. These gatherings are the COP meetings to the Convention on Biological Diversity (CBD). Last year, we wrote about the then forthcoming COP15 to the CBD (Nat. Sustain. 4, 189; 2021), the meeting in which the new conservation targets to be met by 2030 were to be agreed. We highlighted the extent to which experts worried that those new targets might not go far enough. The meeting was postponed more than once due to the COVID-19 pandemic, and it is finally happening on 7 December 2022, in Montreal, Canada. The world has already seen 15 COP meetings to the CBD, how many more meetings will be needed for the biodiversity crisis to be averted?But let’s go back to thinking about sustainability. Experts look for holistic visions of the world. Here is an interesting example of what holism means. Biodiversity decline and climate change are both the result of the broken connection between people and the rest of nature, they ultimately have the same, deep roots. They are mutually reinforcing phenomena: unhealthy biodiversity contributes to climate change, and climate change makes biodiversity ill. All this is bad news for human and planetary well-being. The climate–biodiversity conundrum, at least to some degree, has been recognized at a higher level — during COP27, leaders dedicated one day to biodiversity.Yet, given that these issues are highly interconnected and have the same origin, why is the world insisting on discussing them as separate agendas? Why are we still holding two separate COPs? How are these meetings going to promote any fruitful synergy? How will they lead people to reconnect with the rest of nature? Country representatives should be breaking silos, embracing holism and bringing these intertwined issues, and their multiple ramifications, to the same negotiating table.Nature Sustainability welcomes the long-awaited COP15 to the CBD and hopes that countries will agree on feasible yet ambitious 2030 targets to protect and enhance biodiversity. But most of all, we hope that all of the experts and leaders involved in addressing the environmental crises embrace holism to promote meaningful actions across the world aimed at restoring people’s connection with the rest of nature. We are eager to see progress to this end. In the meantime, the collection we started in March 2021 with Nature Ecology & Evolution has been updated to renew our support to the biodiversity community. More

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    The application of a CART model for forensic human geolocation using stable hydrogen and oxygen isotopes

    The isotopic spread for each study siteThe overall linear relationship between δ2H and δ18O values for hair (n = 81) and toenails (n = 39), respectively, were (Fig. 2):$$delta^{2} {text{H}}_{{text{hair(VSMOW)}}} = , 0.89 times delta^{18} {text{O}}_{{text{hair(VSMOW)}}} {-} , 86.16,;{text{R}}^{2} = , 0.19,;p , < , 0.01$$ (1) $$delta^{2} {text{H}}_{{text{toenail(VSMOW)}}} = , 0.15 times delta^{18} {text{O}}_{{text{toenail(VSMOW)}}} {-} , 91.69,;{text{R}}^{2} = , 0.00,;p , = , 0.69$$ (2) Figure 2δ2H and δ18O values (‰) of all samples for both hair (δ2H: n = 81, δ18O: n = 82) and toenails (δ2H and δ18O: n = 39). The solid black line represents the Global Meteoric Water Line (GMWL) [δ2H = 8 (times) δ18O + 10] and is included in the graph for comparison purposes. The regression lines between oxygen and hydrogen values for hair [δ2Hhair(VSMOW) = 0.89 × δ18Ohair(VSMOW) − 86.16, R2 = 0.19, p  − 82‰ were then split further where any samples with δ2Hhair values less than − 73‰ were initially classified as Site 2. These samples were then split again to either Site 2 (δ2Hhair ≥ 76‰) or Site 4 (δ2Hhair  − 73‰ were classified as Site 4. No samples could be classified as originating from Site 3. The second CART model was built for stable hydrogen and oxygen isotopes of toenails (Model 2) (Fig. 5b). The model included only two decision nodes in which the first predictor variable was δ2Htoenail value, where samples with values less than − 93‰ were predicted to be from Site 1. For toenail samples with hydrogen values greater than − 93‰, oxygen values were used to determine whether they could be classified as Site 2 or Site 4. Those samples with δ18Otoenail values less than 9.6‰ were classified as Site 2 and those with values greater than 9.6‰ were predicted as Site 4. No samples were predicted to be from Site 3 purely from stable hydrogen and oxygen isotopes in toenails. Finally, the third model consisted of stable hydrogen and oxygen isotope values in both hair and toenail samples (Model 3) (Fig. 5c). Model 3 selected toenails as the best attribute for classification, which indicates that toenail isotope values are the better predictor when both hair and toenail samples are present for analysis from Sites 1–4. The model was similar to that of Model 2.Figure 5Decision trees developed from both δ2H and δ18O values of (a) hair [Model 1, trained with n = 65], (b) toenails [Model 2, trained with n = 32] and (c) of both hair and toenails [Model 3, trained with n = 28]. The predicted study site numbers are shown on the first row within each bubble. The proportions of samples in each node are shown as decimals for Sites 1, 2, 3, 4, respectively. The percentages indicate the proportion of samples within each sub-partition.Full size imageConfusion matrices (Table 1) were constructed for all three models to evaluate the performance of the classification models. Of the three models, Model 3 proved to be the most accurate model with an overall accuracy of 71.4% (see Supplementary Fig S2. online). The performance evaluation summary, including measures for sensitivity, specificity, positive predictive value, and negative predictive value for all three models, is provided in (see Supplementary Table S3. online).Intra-individual differencesBoth hair and toenail samples were retrieved from 35 of the 86 individuals. The paired difference between δ2H values in hair and toenails of the same individual was tested using the Wilcoxon Signed Rank's test for non-normal data as the dataset failed the Shapiro–Wilk's normality test at the α = 0.05 significance level. Significant differences were found between δ2H values of hair (n = 35, mean = − 78.0‰, s.d. = 3.06) and toenails (n = 35, mean = − 90.9‰, s.d. = 3.27) from the same individual (p  0.05. Overall, the isotopic values of δ2H in hair were higher than those of toenail from the same individual by 13.0‰, on average, with a standard deviation of 8.4‰. For δ18O, the average was 1.5‰ with a standard deviation of 4.6‰ (Fig. 6).Figure 6(a) δ2H and (b) δ18O values in hair and toenails for all individuals that provided both tissue types (n = 35). Study site information are also shown by shapes. The standard deviations of each sample, ran in either duplicates or triplicates, are shown by error bars. Note that error bars cannot be seen for some samples due to small standard deviations. The average difference between the isotopic values of hair and toenail from the same individual were 13.0‰ with a standard deviation of 8.4‰ for δ2H and 1.5‰ with a standard deviation of 4.6‰ for δ18O.Full size imageThe linear relationships between δ2H in hair and toenails for all individuals were (see Supplementary Fig S3. online):$$delta^{2} {text{H}}_{{{text{hair}}}} = , 0.48 times delta^{2} {text{H}}_{{text{toenail }}} {-} , 34.72,;{text{R}}^{2} = , 0.16,;p , < , 0.05$$ (19) and for δ18O:$$delta^{18} {text{O}}_{{{text{hair}}}} = , 0.55 times delta^{18} {text{O}}_{{{text{toenail}}}} + , 5.16,;{text{R}}^{2} = , 0.13,;p , < 0.05$$ (20) Overall, both equations showed a weak relationship, as seen by the small R2 values. More

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    Author Correction: Adult sex ratios: causes of variation and implications for animal and human societies

    Department of Anthropology, East Carolina University, Greenville, NC, USARyan SchachtDepartment of Environmental Science, Policy and Management and Museum of Vertebrate Zoology, University of California, Berkeley, CA, 94720, USASteven R. BeissingerDepartment of Ecology and Evolution, University of Lausanne, 1015, Lausanne, SwitzerlandClaus WedekindEcology & Evolution, Research School of Biology, The Australian National University, Acton, Canberra, 2601, AustraliaMichael D. JennionsMARBEC Univ Montpellier, CNRS, Ifremer, IRD, Montpellier, FranceBenjamin GeffroyELKH-PE Evolutionary Ecology Research Group, University of Pannonia, 8210, Veszprém, HungaryAndrás LikerBehavioural Ecology Research Group, Center for Natural Sciences, University of Pannonia, 8210, Veszprém, HungaryAndrás LikerBehavioral Ecology and Sociobiology Unit, German Primate Center, Leibniz Institute of Primate Biology, 37077, Göttingen, GermanyPeter M. KappelerDepartment of Sociobiology/Anthropology, University of Göttingen, 37077, Göttingen, GermanyPeter M. KappelerGroningen Institute for Evolutionary Life Sciences, University of Groningen, 9747 AG, Groningen, The NetherlandsFranz J. WeissingDepartment of Anthropology, University of Utah, Salt Lake City, UT, USAKaren L. KramerInstitute of Global Health, University College London, London, UKTherese HeskethCentre for Global Health, Zhejiang University School of Medicine, Hangzhou, P.R. ChinaTherese HeskethIHPE Univ Perpignan Via Domitia, CNRS, Ifremer, Univ Montpellier, Perpignan, FranceJérôme BoissierStockholm University Demography Unit, Sociology Department, Stockholm University, 106 91, Stockholm, SwedenCaroline UgglaKem C. Gardner Policy Institute, David Eccles School of Business, University of Utah, Salt Lake City, UT, USAMike HollingshausMilner Centre for Evolution, University of Bath, Bath, BA2 7AY, UKTamás SzékelyELKH-DE Reproductive Strategies Research Group, Department of Zoology and Human Biology, University of Debrecen, H-4032, Debrecen, HungaryTamás Székely More

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    DNA reveals that mastodons roamed a forested Greenland two million years ago

    RESEARCH BRIEFINGS
    07 December 2022

    Ancient environmental DNA from northern Greenland opens a new chapter in genetic research, demonstrating that it is possible to track the ecology and evolution of biological communities two million years ago. The record shows an open boreal-forest ecosystem inhabited by large animals such as mastodons and reindeer. More

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