More stories

  • in

    Variation in blubber cortisol levels in a recovering humpback whale population inhabiting a rapidly changing environment

    Wikelski, M. & Cooke, S. J. Conservation physiology. Trends Ecol. Evol. 21, 38–46 (2006).Article 
    PubMed 

    Google Scholar 
    Cattet, M. et al. Understanding grizzly bear health in the context of changing landscapes. Foothills Model Forest Grizzly Bear Research Program Annual Report, 80–86 (2005).Reeder, D. M. & Kramer, K. M. Stress in free-ranging mammals: Integrating physiology, ecology, and natural history. J. Mammal. 86, 225–235. https://doi.org/10.1644/bhe-003.1 (2005).Article 

    Google Scholar 
    Dunlop, R. A., Braithwaite, J., Mortensen, L. O. & Harris, C. M. Assessing population-level effects of anthropogenic disturbance on a marine mammal population. Front. Mar. Sci. 8, 230 (2021).Article 

    Google Scholar 
    Atkinson, S., Crocker, D., Houser, D. & Mashburn, K. Stress physiology in marine mammals: How well do they fit the terrestrial model?. J. Comp. Physiol. B. 185, 463–486. https://doi.org/10.1007/s00360-015-0901-0 (2015).Article 
    CAS 
    PubMed 

    Google Scholar 
    Romero, L. M. & Beattie, U. K. Common myths of glucocorticoid function in ecology and conservation. J. Exp. Zool. Part A: Ecol. Integr. Physiol. 337, 7–14. https://doi.org/10.1002/jez.2459 (2022).Article 
    CAS 

    Google Scholar 
    Champagne, C. D. et al. Blubber cortisol qualitatively reflects circulating cortisol concentrations in bottlenose dolphins. Mar. Mamm. Sci. 33, 134–153 (2017).Article 
    CAS 

    Google Scholar 
    Champagne, C. D. et al. Comprehensive endocrine response to acute stress in the bottlenose dolphin from serum, blubber, and feces. Gen. Comp. Endocrinol. 266, 178 (2018).Article 
    CAS 
    PubMed 

    Google Scholar 
    Teerlink, S., Horstmann, L. & Witteveen, B. Humpback whale (Megaptera novaeangliae) blubber steroid hormone concentration to evaluate chronic stress response from whale-watching vessels. Aquat. Mamm. 44, 411 (2018).Article 

    Google Scholar 
    Mingramm, F. M., Keeley, T., Whitworth, D. J. & Dunlop, R. A. Blubber cortisol levels in humpback whales (Megaptera novaeangliae): A measure of physiological stress without effects from sampling. Gen. Comp. Endocrinol. 291, 113436 (2020).Article 
    CAS 
    PubMed 

    Google Scholar 
    Saco, Y. et al. Evaluation of serum cortisol, metabolic parameters, acute phase proteins and faecal corticosterone as indicators of stress in cows. Vet. J. 177, 439–441 (2008).Article 
    CAS 
    PubMed 

    Google Scholar 
    Rolland, R. M. et al. Evidence that ship noise increases stress in right whales. Proc. R. Soc. B: Biol. Sci. 279, 2363–2368. https://doi.org/10.1098/rspb.2011.2429 (2012).Article 

    Google Scholar 
    Rocha, R., Clapham, P. J. & Ivashchenko, Y. V. Emptying the oceans: A summary of industrial whaling catches in the 20th century. Mar. Fish. Rev 76, 37–48 (2014).Article 

    Google Scholar 
    Comission, I. W. Report of the scientific committee. Journal of Cetacean Research and Management SC/68C (2021).Ducklow, H. W. et al. West Antarctic Peninsula: An ice-dependent coastal marine ecosystem in transition. Oceanography 26, 190–203 (2013).Article 

    Google Scholar 
    Laws, R. Seals and whales of the Southern Ocean 81–96 (Philosophical Transactions of the Royal Society of London. Series B, 1977).
    Google Scholar 
    Savoca, M. S. et al. Baleen whale prey consumption based on high-resolution foraging measurements. Nature 599, 85–90. https://doi.org/10.1038/s41586-021-03991-5 (2021).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Pallin, L. J. et al. High pregnancy rates in humpback whales (Megaptera novaeangliae) around the Western Antarctic Peninsula, evidence of a rapidly growing population. R. Soc. Open Sci. https://doi.org/10.1098/rsos.180017 (2018).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Bender, N. A., Crosbie, K. & Lynch, H. J. Patterns of tourism in the Antarctic Peninsula region: A 20-year analysis. Antarct. Sci. 28, 194–203. https://doi.org/10.1017/s0954102016000031 (2016).Article 
    ADS 

    Google Scholar 
    Operators, I. A. o. A. T. (2019).Trumble, S. J. et al. Baleen whale cortisol levels reveal a physiological response to 20th century whaling. Nat. Commun. https://doi.org/10.1038/s41467-018-07044-w (2018).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Cates, K. A. et al. Corticosterone in central north pacific male humpback whales (Megaptera novaeangliae): Pairing sighting histories with endocrine markers to assess stress. Gen. Comp. Endocrinol. 296, 113540 (2020).Article 
    CAS 
    PubMed 

    Google Scholar 
    Sprogis, K. R., Videsen, S. & Madsen, P. T. Vessel noise levels drive behavioural responses of humpback whales with implications for whale-watching. eLife https://doi.org/10.7554/elife.56760 (2020).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Rolland, R. et al. Fecal glucocorticoids and anthropogenic injury and mortality in North Atlantic right whales Eubalaena glacialis. Endanger. Species Res. 34, 417–429. https://doi.org/10.3354/esr00866 (2017).Article 

    Google Scholar 
    Modest, M. et al. First description of migratory behavior of humpback whales from an Antarctic feeding ground to a tropical calving ground. Anim. Biotelem. 9, 1–16 (2021).Article 

    Google Scholar 
    Amaral, R. S. Use of alternative matrices to monitor steroid hormones in aquatic mammals: A review. Aquat. Mamm. 36, 162 (2010).Article 

    Google Scholar 
    Graham, K. M., Burgess, E. A. & Rolland, R. M. Stress and reproductive events detected in North Atlantic right whale blubber using a simplified hormone extraction protocol. Conserv. Physiol. https://doi.org/10.1093/conphys/coaa133 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Atkinson, S. et al. Pregnancy rate and biomarker validations from the blubber of eastern North Pacific blue whales. Mar. Mamm. Sci. https://doi.org/10.1111/mms.12616 (2019).Article 

    Google Scholar 
    Hunt, K. E., Rolland, R. M., Kraus, S. D. & Wasser, S. K. Analysis of fecal glucocorticoids in the North Atlantic right whale (Eubalaena glacialis). Gen. Comp. Endocrinol. 148, 260–272. https://doi.org/10.1016/j.ygcen.2006.03.012 (2006).Article 
    CAS 
    PubMed 

    Google Scholar 
    Mashburn, K. L. & Atkinson, S. Evaluation of adrenal function in serum and feces of Steller sea lions (Eumetopias jubatus): Influences of molt, gender, sample storage, and age on glucocorticoid metabolism. Gen. Comp. Endocrinol. 136, 371–381. https://doi.org/10.1016/j.ygcen.2004.01.016 (2004).Article 
    CAS 
    PubMed 

    Google Scholar 
    Jeanniard Du Dot, T. et al. Changes in glucocorticoids, IGF-I and thyroid hormones as indicators of nutritional stress and subsequent refeeding in Steller sea lions (Eumetopias jubatus). Comp. Biochem. Physiol. Part A Mol. Integr. Physiol 152, 524–534. https://doi.org/10.1016/j.cbpa.2008.12.010 (2009).Article 
    CAS 

    Google Scholar 
    Foley, C. A. H., Papageorge, S. & Wasser, S. K. Noninvasive stress and reproductive measures of social and ecological pressures in free-ranging African elephants. Conserv. Biol. 15, 1134–1142. https://doi.org/10.1046/j.1523-1739.2001.0150041134.x (2001).Article 

    Google Scholar 
    Challis, J. R., Matthews, S. G., Gibb, W. & Lye, S. J. Endocrine and paracrine regulation of birth at term and preterm. Endocr. Rev. 21, 514–550 (2000).CAS 

    Google Scholar 
    Robeck, T. R., Steinman, K. J. & O’Brien, J. K. Characterization and longitudinal monitoring of serum androgens and glucocorticoids during normal pregnancy in the killer whale (Orcinus orca). Gen. Comp. Endocrinol. 247, 116–129 (2017).Article 
    CAS 
    PubMed 

    Google Scholar 
    Rolland, R. M., Hunt, K. E., Kraus, S. D. & Wasser, S. K. Assessing reproductive status of right whales (Eubalaena glacialis) using fecal hormone metabolites. Gen. Comp. Endocrinol. 142, 308–317 (2005).Article 
    CAS 
    PubMed 

    Google Scholar 
    Burgess, E. A., Hunt, K. E., Kraus, S. D. & Rolland, R. M. Adrenal responses of large whales: Integrating fecal aldosterone as a complementary biomarker to glucocorticoids. Gen. Comp. Endocrinol. 252, 103–110. https://doi.org/10.1016/j.ygcen.2017.07.026 (2017).Article 
    CAS 
    PubMed 

    Google Scholar 
    Ducklow, H. W. et al. Marine pelagic ecosystems: The west Antarctic Peninsula. Philos. Trans. R. Soc. London B: Biol. Sci. 362, 67–94 (2007).Article 
    PubMed 

    Google Scholar 
    Rogers, A. et al. Antarctic futures: An assessment of climate-driven changes in ecosystem structure, function, and service provisioning in the Southern Ocean. Ann. Rev. Mar. Sci. 12, 87–120 (2020).Article 
    CAS 
    PubMed 

    Google Scholar 
    Hillbrand, F. & Elsaesser, F. Concentrations of progesterone in the backfat of pigs during the oestrous cycle and after ovariectomy. J. Reprod. Fertil. 69, 73–80 (1983).Article 
    CAS 
    PubMed 

    Google Scholar 
    Funasaka, N. et al. Seasonal difference of diurnal variations in serum melatonin, cortisol, testosterone, and rectal temperature in Indo-Pacific bottlenose dolphins (Tursiops aduncus). Aquat. Mamm. 37, 433 (2011).Article 

    Google Scholar 
    Oki, C. & Atkinson, S. Diurnal patterns of cortisol and thyroid hormones in the Harbor seal (Phoca vitulina) during summer and winter seasons. Gen. Comp. Endocrinol. 136, 289–297. https://doi.org/10.1016/j.ygcen.2004.01.007 (2004).Article 
    CAS 
    PubMed 

    Google Scholar 
    Lavigne, D., Innes, S., Worthy, G. & Edwards, E. F. Lower critical temperatures of blue whales, Balaenoptera musculus. J. Theor. Biol. 144, 249–257 (1990).Article 
    ADS 

    Google Scholar 
    Nichols, R. C. et al. Intra-seasonal variation in feeding rates and diel foraging behaviour in a seasonally fasting mammal, the humpback whale. R. Soc. Open Sci. https://doi.org/10.1098/rsos.211674 (2022).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Bierlich, K. C. Incorporating Photogrammetric Uncertainty in UAS-based Morphometric Measurements of Baleen Whales, (2021).Kellar, N. M. et al. Blubber cortisol: A potential tool for assessing stress response in free-ranging dolphins without effects due to sampling. PLoS ONE 10, e0115257 (2015).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Bejder, L. & Samuels, A. Evaluating the effects of nature-based tourism on cetaceans. Mar. Mamm. Fish. Tour. Manag. Issues 1, 229–256 (2003).
    Google Scholar 
    New, L. F. et al. The modelling and assessment of whale-watching impacts. Ocean Coast. Manag. 115, 10–16 (2015).Article 

    Google Scholar 
    Avila, I. C., Correa, L. M. & Parsons, E. Whale-watching activity in Bahía Málaga, on the Pacific coast of Colombia, and its effect on humpback whale (Megaptera novaeangliae) behavior. Tour. Mar. Environ. 11, 19–32 (2015).Article 

    Google Scholar 
    Amrein, A. M., Guzman, H. M., Surrey, K. C., Polidoro, B. & Gerber, L. R. Impacts of whale watching on the behavior of humpback whales (Megaptera novaeangliae) in the Coast of Panama. Front. Mar. Sci. 7, 1105 (2020).Article 

    Google Scholar 
    Heenehan, H. et al. Caribbean Sea soundscapes: monitoring humpback whales, biological sounds, geological events, and anthropogenic impacts of vessel noise. Front. Mar. Sci., 347 (2019).Keay, J. M., Singh, J., Gaunt, M. C. & Kaur, T. Fecal glucocorticoids and their metabolites as indicators of stress in various mammalian species: A literature review. J. Zoo Wildl. Med. 37, 234–244 (2006).Article 
    PubMed 

    Google Scholar 
    Harris, K., Gende, S. M., Logsdon, M. G. & Klinger, T. Spatial pattern analysis of cruise ship-humpback whale interactions in and near Glacier Bay National Park, Alaska. Environ. Manag. 49, 44–54. https://doi.org/10.1007/s00267-011-9754-9 (2012).Article 
    ADS 

    Google Scholar 
    Palsbøll, P. J., Larsen, F. & Hansen, E. S. Sampling of skin biopsies from free-raging large cetaceans in west greenland: Development of new biopsy tips and bolt designs. International Whaling Commission Special Issue Series (1991).Weinstein, B. G., Double, M., Gales, N., Johnston, D. W. & Friedlaender, A. S. Identifying overlap between humpback whale foraging grounds and the Antarctic krill fishery. Biol. Cons. 210, 184–191 (2017).Article 

    Google Scholar 
    Lambertsen, R. H. A biopsy system for large whales and its use for cytogenetics. J. Mammal. 68, 443–445. https://doi.org/10.2307/1381495 (1987).Article 

    Google Scholar 
    Gilson, A., Syvanen, M., Levine, K. & Banks, J. Deer gender determination by polymerase chain reaction. Calif. Fish Game 84, 159–169 (1998).
    Google Scholar 
    Aasen, E. & Medrano, J. F. Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats. Bio/Technology 8, 1279–1281 (1990).CAS 
    PubMed 

    Google Scholar 
    Valsecchi, E. & Amos, W. Microsatellite markers for the study of cetacean populations. Mol. Ecol. 5, 151–156 (1996).Article 
    CAS 
    PubMed 

    Google Scholar 
    Palsbøll, P., Bérubé, M., Larsen, A. & Jørgensen, H. Primers for the amplification of tri-and tetramer microsatellite loci in baleen whales. Mol. Ecol. 6, 893–895 (1997).Article 
    PubMed 

    Google Scholar 
    Berube, M., Jørgensen, H., McEwing, R. & Palsbøll, P. J. Polymorphic di-nucleotide microsatellite loci isolated from the humpback whale, Megaptera novaeangliae. Mol. Ecol. 9, 2181–2183 (2000).Article 
    CAS 
    PubMed 

    Google Scholar 
    Waldick, R., Brown, M. & White, B. Characterization and isolation of microsatellite loci from the endangered North Atlantic right whale. Mol. Ecol. 8, 1763–1765 (1999).Article 
    CAS 
    PubMed 

    Google Scholar 
    Baker, C. S. et al. Strong maternal fidelity and natal philopatry shape genetic structure in North Pacific humpback whales. Mar. Ecol. Progress Ser. 494, 291 (2013).Article 
    ADS 

    Google Scholar 
    Constantine, R. et al. Abundance of humpback whales in Oceania using photo-identification and microsatellite genotyping. Mar. Ecol. Prog. Ser. 453, 249–261 (2012).Article 
    ADS 

    Google Scholar 
    Peakall, R. & Smouse, P. E. GENALEX 6: Genetic analysis in Excel. Population genetic software for teaching and research. Mol. Ecol. Notes 6, 288–295 (2006).Article 

    Google Scholar 
    Kalinowski, S. T., Taper, M. L. & Marshall, T. C. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Mol. Ecol. 16, 1099–1106 (2007).Article 
    PubMed 

    Google Scholar 
    Kellar, N. M., Trego, M. L., Marks, C. I. & Dizon, A. E. Determining pregnancy from blubber in three species of delphinids. Mar. Mamm. Sci. 22, 1–16 (2006).Article 

    Google Scholar 
    Pallin, L., Robbins, J., Kellar, N., Bérubé, M. & Friedlaender, A. Validation of a blubber-based endocrine pregnancy test for humpback whales. Conserv. Physiol. https://doi.org/10.1093/conphys/coy031 (2018).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Kellar, N. M. et al. Low reproductive success rates of common bottlenose dolphins Tursiops truncatus in the northern Gulf of Mexico following the Deepwater Horizon disaster (2010–2015). Endanger. Species Res. 33, 143–158 (2017).Article 

    Google Scholar 
    R: A language and environment for statistical computing. (R Foundation for Statistical Computing, Vienna, Austria, 2021).ArcMap (version 10.8.2) (Redlands, CA: Esri Inc, 2022). More

  • in

    Intracellular common gardens reveal niche differentiation in transposable element community during bacterial adaptive evolution

    Bacterial strains, primers, and growth conditionsBacterial strains, plasmids, and primers used in this study are shown in Supplementary Table S1. Escherichia coli strains carrying plasmids used in conjugation experiments were grown at 37 °C in LB medium. S. fredii CCBAU25509 (SF2) and its derivatives were grown at 28 °C in TY medium (5 g tryptone, 3 g yeast extract, 0.6 g CaCl2 per liter). To screen and purify conjugants or obtain pure cultures of bacteria, antibiotics were supplemented as required at the following concentrations (μg/mL): for E. coli, gentamicin (Gen), 30; and kanamycin (Km), 100; for Sinorhizobium strains, trimethoprim (Tmp), 10; nalidixic acid (Na), 30; and kanamycin (Km), 100. To screen sacB mutants from SF2 derivatives, firstly SF2 tolerance of 8%-30% sucrose in the TY medium was measured by the growth curve using Bioscreen C (Oy Growth Curves Ab Ltd, Raisio, Finland), and then the TY medium containing 10% sucrose was chosen as the selection medium.Construction of S. fredii derivatives harboring xenogeneic PsacB-sacB
    The multipartite genome of SF2 consists of a chromosome (Ch, GC% = 62.6%), a chromid (pB, GC% = 62%) [31], and a symbiosis plasmid (pA, GC% = 59%) [26]. Within each replicon, an insertion position, with GC% of its 10 kb flanking region being the same as the replicon average, was chosen for subsequent experiments (Fig. 1A). The suicide plasmid pJQ200SK carries the wild-type sacB gene (characterized by its low GC content of 38.8%; 1422 bp) and its promoter region PsacB (GC% = 36.1%, 446 bp) from Bacillus subtilis subsp. subtilis str. 168 [32]. A Km-resistant cassette from pBBR1MCS-2 [33] was amplified and assembled with a linearized pJQ200SK lacking the Gm-resistant cassette using a seamless cloning kit (Taihe Biotechnology, Beijing, China) as described previously [34]. This generated pJQ-L carrying the wild-type low GC% sacB (38.8%; 1422 bp; L-GC). The sacB gene with medium (54.6%; M-GC) or high GC (61.6%; H-GC) content in its synonymous codons was synthesized (Fig. S1), and used to replace the wild-type low GC% sacB gene of pJQ-L to generate pJQ-M and pJQ-H. This was also performed using the seamless cloning method as described above with the linearized pJQ-L lacking the wild-type sacB. Three genomic segments of SF2 (pA:330682-331687, pB:702541-703493, Ch:674057-675207) were individually cloned into each of pJQ-L, pJQ-M, and pJQ-H at the SmaI site using the seamless cloning method, which allowed subsequent integration of xenogeneic cassettes into three replicons. This generated nine plasmids (pJQ-L_pA, pJQ-L_pB, pJQ-L_Ch; pJQ-M_pA, pJQ-M_pB, pJQ-M_Ch; pJQ-H_pA, pJQ-H_pB, pJQ-H_Ch), which were transformed into E. coli DH5α and verified by Sanger sequencing before conjugation into rhizobia via triparental mating with helper plasmid pRK2013 [35]. This generated nine SF2 derivatives individually carrying a xenogeneic cassette in a replicon (Fig. 1A). The correct insertion of the xenogeneic cassette was checked by PCR.Fig. 1: Screening mutations in xenogeneic sacB of different GC content.A The xenogeneic cassettes harboring sacB of L-GC, M-GC, or H-GC were individually inserted into the symbiosis plasmid (pA; GC% = 59%), chromid (pB; GC% = 62%), or chromosome (Ch; GC% = 62.6%) of Sinorhizobium fredii CCBAU25509. Gene IDs surrounding each insertion position are shown. GC% of the three sacB versions were 38.8% (L-GC, the wild-type version from Bacillus subtilis subsp. subtilis str. 168), 54.6% (M-GC, synthesized), and 61.6% (H-GC, synthesized). The wild-type PsacB (GC% = 36.1%, 446 bp) of B. subtilis 168 was cloned together with each of the three versions of sacB. The number of A, T, C, or G in the 1422 bp sacB gene is indicated. B Growth curves in TY medium. C Levansucrase enzyme activity assay of crude proteins collected at OD600 = 1.2 in TY medium. Different letters indicate significant difference (Average ± SEM; ANOVA followed by Duncan’s test, alpha = 0.05). D Growth curves in TY medium supplemented with 10% sucrose. E Schematic view of culturing, mutant screening, and mutation identification in this work. sacB, levansucrase gene; km, kanamycin resistance gene.Full size imageThe xenogeneic silencer MucR prefers low GC% DNA targets [29, 30], and its potential role in niche differentiation for IS community members was tested. SF2 has two mucR copies, and the in-frame deletion mutant ΔmucR1R2 was constructed by using an allelic exchange strategy: upstream and downstream ~500 bp flanking regions of mucR1 or mucR2 were amplified and assembled with the linearized allelic exchange vector pJQ200SK. The pJQ200SK derivative used to delete mucR1 was linearized and then cloned seamlessly with the sequence coding MucR1 and C-terminal fused FLAG-tag. The resultant plasmid was conjugated into SF2 to generate SF2MucR1FLAG. The xenogeneic cassettes carrying plasmids (pJQ-L_pA, pJQ-M_pA, pJQ-H_pA) were then inserted into the same position of pA in ΔmucR1R2 and SF2MucR1FLAG, and verified by PCR.Mutant screening and calculation of mutation frequencyTo screen sacB mutants from SF2 derivatives, single colonies of S. fredii derivatives were inoculated and grown to an OD600 = 0.2, 0.6, 1.2, and 2.0, and dilutions were applied to plates with and without 10% sucrose respectively. The number of colonies on the 10% sucrose TY plates was recorded as “A” at the dilution of 10−a, and the number of colonies on the sucrose-free TY plates was recorded as “B” at the dilution of 10−b. The total mutation frequency was then calculated by (A·10-a)/(B·10-b). Independent colonies on the 10% sucrose TY plates were further purified on the same medium plates, and the full length of PsacB-sacB fragment was amplified by colony PCR. Gene loss, SNPs or short InDels, or large insertion mutations were identified by electrophoresis analysis of PCR products. Representative clones with large insertion mutations were selected for Sanger sequencing. Three independent experiments were performed for all test strains.Enzyme activity assay for levansucraseTo evaluate the function of xenogeneic sacB in SF2 derivatives, sucrose was dissolved in the buffer solution (0.1 M CH3COONa, pH 5.5), and the total protein extract of bacteria was added (calibrated to the same concentration) to make the final concentration of sucrose 1%, and the reaction system was incubated at 28°C for 12 h. After adding the color development solution (3,5-dinitrosalicylic acid 6.3 g, sodium hydroxide 21.0 g, potassium sodium tartrate 182.0 g, phenol 5.0 g, sodium metabisulfite 5.0 g in 1000 mL water; BOXBIO, Beijing, China), the enzyme was inactivated at 95 °C for 5 min, and the absorbance value at 540 nm was measured to calculate the glucose content. Determination of the release of glucose and fructose from sucrose allowed calculation of the total activity of the levansucrase. One unit (U) of enzyme is defined as the amount of enzyme required for producing 1 µmol glucose per min in reaction buffer. The specific activity of levansucrase hydrolysis activity is the activity units per mg of protein (U/mg).5′RACETo determine the transcription start site of the sacB gene, a 5′RACE experiment was performed with the 5′RACE kit (Sangon, Beijing, China) for Rapid Amplification of cDNA Ends using three gene-specific primers (Table S1) that anneal to the known region and an adapter primer that targets the 5′ end. Products generated by 5′RACE were subcloned into the TOPO-TA vector and individual colonies were sequenced.RNA extraction and RT-qPCRTo determine transcriptional levels of the major active ISs in SF2 and its ΔmucR1R2 mutant, strains were grown in 50 mL TY liquid medium to an OD600 of 1.2. A bacterial total RNA Kit (Zomanbio, Beijing, China) was used for total RNA extraction. cDNA was synthesized using FastKing-RT SuperMix (TIANGEN, Beijing, China). qPCR was performed by using QuantStudio 6 Flex and 2× RealStar Green Mixture (Genstar, Beijing, China). The primer pairs used are listed in Table S1. The 16S rRNA gene was used as an internal reference to normalize the expression level. Three independent biological replicates were performed.ChIP-qPCRTo test the potential recruitment of MucR in the xenogeneic PsacB-sacB region, three SF2 derivative strains harboring sacB of different GC% in the pA replicon and MucR1-FLAG (Table S1; MucR1-FLAG: L-GC, MucR1-FLAG: M-GC, MucR1-FLAG: H-GC) were cultured until the OD600 had reached 1.2. Formaldehyde was added into the TY medium to a final concentration of 1%, which was then incubated at 28 °C for 15 min. To stop crosslinking, glycine was added to a final concentration of 0.1 M. The cross-linked samples were harvested (5000 × g, 5 min, 4 °C) and washed twice with cold phosphate-buffered saline (PBS). After the pellets were ground into fine powder in liquid nitrogen, the samples were resuspended in buffer containing 1% SDS and 1 mM phenylmethanesulfonyl fluoride, and lysed by sonication using a sonicator (Q800R3, QSonica). Chromatin immunoprecipitation (ChIP) was performed using the ChIP assay kit (Beyotime, Shanghai, China) according to the manufacturer’s recommendations. The supernatant was collected and chromatin was immunoprecipitated with Anti-FLAG M2 antibody (Sigma). Input control and DNA obtained from the immunoprecipitation were amplified by PCR using primers listed in Table S1. The recruitment level of FLAG-tagged MucR1 in multiple regions within the PsacB-sacB fragment inserted by ISs at high frequency was detected by ChIP-qPCR.Crosslinking and western blotting assayTo test the ability of MucR1 to form homodimer in SF2 derivatives carrying sacB in pA, rhizobial cells (SF2MucR1FLAG, MucR1-FLAG: L-GC, MucR1-FLAG: M-GC, and MucR1-FLAG: H-GC) were cultured in 50 mL TY medium to an OD600 of 1.2. Formaldehyde was added at a final concentration of 1% in the culture which was then shaken at 28 °C, 100 rpm for 15 min to allow crosslinking. The crosslinking reaction was terminated by adding a final concentration of 100 mM glycine (28 °C, 100 rpm, 5 min). 1 mL of the above solution was centrifuged (5000 × g, 4 °C, 1 min), resuspended in 50 µL SDS loading buffer to a uniform cell density, and then boiled for 10 minutes for lysis. Next, lysates were separated on 12% SDS-PAGE and transferred to a nitrocellulose membrane. For immunodetection of individual proteins, the method described previously was used [30]. Briefly, mouse monoclonal Anti-FLAG M2 antibody (Sigma), HRP (horseradish peroxidase) conjugated goat Anti-mouse IgG (Abcam), and eECL Western blot kit (CWBIO, Beijing, China) were used, and chemiluminescence signals were visualized using Fusion FX6 (Vilber) and Evolution-Capt Edge software.Protein purificationTo purify MucR1 protein, E. coli BL21(DE3) carrying His6-SUMO-tagged MucR1 in the pET30a [29] was cultured in 500 mL LB medium until OD600 reached 0.8. The procedure described previously was used [30]. IPTG was then added to the culture to a final concentration of 0.6 mM and switched to 18 °C at 150 rpm for 12 h. Cells were harvested by centrifugation (5000 × g, 5 min, 4 °C) and resuspended in 30 mL of lysis buffer (25 mM Tris, pH 8.0, 250 mM NaCl, 10 mM imidazole) supplemented with 0.1 mg/mL DNase I, 0.4 mg/mL of lysozyme, and protease inhibitor mixture (Roche). After 30 min incubation and 120 sonication cycles (300 W, 10 s on, 10 s off), lysates were removed by centrifugation (18,000 × g, 4 °C, 30 min) and filtration through a 0.22 μm membrane. The supernatant was loaded onto Ni-Agarose Resin (CWBIO, Beijing, China) pre-washed using lysis buffer, washed 3 times with wash buffer (lysis buffer containing 20 mM imidazole), and then eluted by lysis buffer containing imidazole gradient (100, 200, 300 mM imidazole). The purified proteins were finally concentrated by ultrafiltration and redissolved in storage buffer (25 mM Tris, pH 8.0, 250 mM NaCl, 10% glycerol) prior to use or storage at −80 °C.DNA bridging assayTo determine if MucR1 can form DNA-MucR1-DNA complex with various regions of xenogeneic PsacB-sacB fragment, a DNA bridging assay described earlier [30, 36] was performed with modifications. DNA probes were prepared by annealing of synthesized complementary strands (PsacB −90~−24) or by PCR amplification (PsacB −90~+3, sacB +710~+802, sacB +908~+1007) using 5′-biotin-labeled or 5′-Cy5 primers (Table S1). In each bridging assay, 100 μL of hydrophilic streptavidin magnetic beads (NEB) were washed twice with 500 μL of PBS and then resuspended in 500 μL of coupling buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 500 mM NaCl). Then, the suspension was supplied with 10 pmol of biotin-labeled DNA and incubated with the beads for 30 min at room temperature with gentle rotation. The resulting beads were washed twice with 500 μL of incubation buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 5% glycerol (vol/vol), 0.05% Tween 20) and resuspended after the addition of 10 pmol Cy5-labeled DNA and 10 μL HRV 3C protease to a final volume of 500 μL. The HRV 3C protease was used herein to remove SUMO. A twofold serial dilution of the protein sample was added to each 50 μL aliquot of bead suspension, and supplemented with incubation buffer to 60 μL final volume. After 30 minutes of incubation with gentle rotation at room temperature, the mixture was placed on a magnetic stand for 5 minutes. The supernatant was collected and labeled as Sample A. The beads were mixed with 60 μL of elution buffer (incubation buffer with 0.1% SDS and 20 μg/mL biotin) and incubated in a boiling water bath for 10 min. The eluted samples were labeled as Sample B. Cy5 fluorescence signals of Sample A and B were detected by a Microscale Thermophoresis Monolith NT.115 system (NanoTemper). The Cy5 fluorescence signal of the Sample A from the treatment without MucR1 was defined as 100% input signal.Statistical analysesAnalysis of variance (ANOVA) followed by Duncan’s test, Student’s t-test, and Fisher’s exact test were performed using GraphPad Prism 8. The closest homolog of individual active ISs and their family identification were determined using ISfinder [37]. Target sequence logos of ISs were generated by multiple sequence alignments of insertion sites within xenogeneic PsacB-sacB or genomic background using the program WebLogo [38].Although the fundamental niche, not constrained by biological interactions, cannot be determined by observation [15], the realized niche, representing a proportion of the fundamental niche where organisms actually live under abiotic and biotic interactions, can be estimated by correlative approaches [15, 39]. In order to address the influence of intracellular variables on biased IS insertions into nine common gardens, the within outlying mean index analysis developed for niche differentiation analysis was carried out using the R package “subniche” [40, 41]. The intracellular environmental gradients were determined by Principal Component Analysis (PCA) based on variables as follows: GC% of different sacB versions, replicon GC%, the number of each IS in the corresponding replicon where sacB is inserted, available insertion sites of ISs in different sacB versions, and levansucrase activity of strains carrying different sacB versions. Within this multidimensional Euclidean space (environmental space), mean positions in realized (sub)niches and parameters of each IS were obtained for the whole data set (realized niches in environmental space defined by nine common gardens) or various subsets (realized subniches in sub-environmental spaces identified by the hierarchical clustering analysis with the ward.D method based on the Euclidean distance matrix) [41]. Two and three subsets rather than four and more subsets were statistically analyzable. By comparing to the overall average habitat conditions (G) or the average subset habitat conditions (GK) of the spatial domain, ISs selecting for a less common habitat were indicated by their significantly higher niche marginality values compared to the simulated values, based on a Monte Carlo test with 1,000 permutations, under the hypothesis that each IS is indifferent to its intracellular environment [40]. More

  • in

    Newer roots for agriculture

    Annual grains, domesticated from wild species, have dominated agriculture since the Neolithic. A new study reports how turning to high-yield perennial rice crops could maintain key ecosystem functions while supporting livelihoods.The past several decades have seen modest but growing investments in the development of perennial grain crops, including perennial counterparts of wheat, rice and sorghum suitable for the USA, China, Europe and Africa. One technique involves domesticating wild perennial species through continual selection of desirable traits over multiple generations3. A recently developed perennial grain currently grown for niche markets in the USA, Kernza, was domesticated from Thinopyrum intermedium, a wild relative of wheat. While yields of Kernza remain low compared with those of annual wheat, they are increasing. As with the development of perennial rice, plant breeders can also cross perennial species with domesticated annual relatives to produce perennial hybrids with desirable traits derived from the annual parent3. More

  • in

    Off the hook: electrical device keeps sharks away from fishing lines

    .readcube-buybox { display: none !important;}
    More than 30% of shark and ray species are edging towards extinction, mainly because they are unintentionally caught by fishers targeting tuna and other commercially valuable species. A new device might help to keep some of these threatened species away from fishing hooks.

    Access options

    /* style specs start */
    style{display:none!important}.LiveAreaSection-193358632 *{align-content:stretch;align-items:stretch;align-self:auto;animation-delay:0s;animation-direction:normal;animation-duration:0s;animation-fill-mode:none;animation-iteration-count:1;animation-name:none;animation-play-state:running;animation-timing-function:ease;azimuth:center;backface-visibility:visible;background-attachment:scroll;background-blend-mode:normal;background-clip:borderBox;background-color:transparent;background-image:none;background-origin:paddingBox;background-position:0 0;background-repeat:repeat;background-size:auto auto;block-size:auto;border-block-end-color:currentcolor;border-block-end-style:none;border-block-end-width:medium;border-block-start-color:currentcolor;border-block-start-style:none;border-block-start-width:medium;border-bottom-color:currentcolor;border-bottom-left-radius:0;border-bottom-right-radius:0;border-bottom-style:none;border-bottom-width:medium;border-collapse:separate;border-image-outset:0s;border-image-repeat:stretch;border-image-slice:100%;border-image-source:none;border-image-width:1;border-inline-end-color:currentcolor;border-inline-end-style:none;border-inline-end-width:medium;border-inline-start-color:currentcolor;border-inline-start-style:none;border-inline-start-width:medium;border-left-color:currentcolor;border-left-style:none;border-left-width:medium;border-right-color:currentcolor;border-right-style:none;border-right-width:medium;border-spacing:0;border-top-color:currentcolor;border-top-left-radius:0;border-top-right-radius:0;border-top-style:none;border-top-width:medium;bottom:auto;box-decoration-break:slice;box-shadow:none;box-sizing:border-box;break-after:auto;break-before:auto;break-inside:auto;caption-side:top;caret-color:auto;clear:none;clip:auto;clip-path:none;color:initial;column-count:auto;column-fill:balance;column-gap:normal;column-rule-color:currentcolor;column-rule-style:none;column-rule-width:medium;column-span:none;column-width:auto;content:normal;counter-increment:none;counter-reset:none;cursor:auto;display:inline;empty-cells:show;filter:none;flex-basis:auto;flex-direction:row;flex-grow:0;flex-shrink:1;flex-wrap:nowrap;float:none;font-family:initial;font-feature-settings:normal;font-kerning:auto;font-language-override:normal;font-size:medium;font-size-adjust:none;font-stretch:normal;font-style:normal;font-synthesis:weight style;font-variant:normal;font-variant-alternates:normal;font-variant-caps:normal;font-variant-east-asian:normal;font-variant-ligatures:normal;font-variant-numeric:normal;font-variant-position:normal;font-weight:400;grid-auto-columns:auto;grid-auto-flow:row;grid-auto-rows:auto;grid-column-end:auto;grid-column-gap:0;grid-column-start:auto;grid-row-end:auto;grid-row-gap:0;grid-row-start:auto;grid-template-areas:none;grid-template-columns:none;grid-template-rows:none;height:auto;hyphens:manual;image-orientation:0deg;image-rendering:auto;image-resolution:1dppx;ime-mode:auto;inline-size:auto;isolation:auto;justify-content:flexStart;left:auto;letter-spacing:normal;line-break:auto;line-height:normal;list-style-image:none;list-style-position:outside;list-style-type:disc;margin-block-end:0;margin-block-start:0;margin-bottom:0;margin-inline-end:0;margin-inline-start:0;margin-left:0;margin-right:0;margin-top:0;mask-clip:borderBox;mask-composite:add;mask-image:none;mask-mode:matchSource;mask-origin:borderBox;mask-position:0 0;mask-repeat:repeat;mask-size:auto;mask-type:luminance;max-height:none;max-width:none;min-block-size:0;min-height:0;min-inline-size:0;min-width:0;mix-blend-mode:normal;object-fit:fill;object-position:50% 50%;offset-block-end:auto;offset-block-start:auto;offset-inline-end:auto;offset-inline-start:auto;opacity:1;order:0;orphans:2;outline-color:initial;outline-offset:0;outline-style:none;outline-width:medium;overflow:visible;overflow-wrap:normal;overflow-x:visible;overflow-y:visible;padding-block-end:0;padding-block-start:0;padding-bottom:0;padding-inline-end:0;padding-inline-start:0;padding-left:0;padding-right:0;padding-top:0;page-break-after:auto;page-break-before:auto;page-break-inside:auto;perspective:none;perspective-origin:50% 50%;pointer-events:auto;position:static;quotes:initial;resize:none;right:auto;ruby-align:spaceAround;ruby-merge:separate;ruby-position:over;scroll-behavior:auto;scroll-snap-coordinate:none;scroll-snap-destination:0 0;scroll-snap-points-x:none;scroll-snap-points-y:none;scroll-snap-type:none;shape-image-threshold:0;shape-margin:0;shape-outside:none;tab-size:8;table-layout:auto;text-align:initial;text-align-last:auto;text-combine-upright:none;text-decoration-color:currentcolor;text-decoration-line:none;text-decoration-style:solid;text-emphasis-color:currentcolor;text-emphasis-position:over right;text-emphasis-style:none;text-indent:0;text-justify:auto;text-orientation:mixed;text-overflow:clip;text-rendering:auto;text-shadow:none;text-transform:none;text-underline-position:auto;top:auto;touch-action:auto;transform:none;transform-box:borderBox;transform-origin:50% 50%0;transform-style:flat;transition-delay:0s;transition-duration:0s;transition-property:all;transition-timing-function:ease;vertical-align:baseline;visibility:visible;white-space:normal;widows:2;width:auto;will-change:auto;word-break:normal;word-spacing:normal;word-wrap:normal;writing-mode:horizontalTb;z-index:auto;-webkit-appearance:none;-moz-appearance:none;-ms-appearance:none;appearance:none;margin:0}.LiveAreaSection-193358632{width:100%}.LiveAreaSection-193358632 .login-option-buybox{display:block;width:100%;font-size:17px;line-height:30px;color:#222;padding-top:30px;font-family:Harding,Palatino,serif}.LiveAreaSection-193358632 .additional-access-options{display:block;font-weight:700;font-size:17px;line-height:30px;color:#222;font-family:Harding,Palatino,serif}.LiveAreaSection-193358632 .additional-login >li:not(:first-child)::before{transform:translateY(-50%);content:””;height:1rem;position:absolute;top:50%;left:0;border-left:2px solid #999}.LiveAreaSection-193358632 .additional-login >li:not(:first-child){padding-left:10px}.LiveAreaSection-193358632 .additional-login >li{display:inline-block;position:relative;vertical-align:middle;padding-right:10px}.BuyBoxSection-683559780{display:flex;flex-wrap:wrap;flex:1;flex-direction:row-reverse;margin:-30px -15px 0}.BuyBoxSection-683559780 .box-inner{width:100%;height:100%}.BuyBoxSection-683559780 .readcube-buybox{background-color:#f3f3f3;flex-shrink:1;flex-grow:1;flex-basis:255px;background-clip:content-box;padding:0 15px;margin-top:30px}.BuyBoxSection-683559780 .subscribe-buybox{background-color:#f3f3f3;flex-shrink:1;flex-grow:4;flex-basis:300px;background-clip:content-box;padding:0 15px;margin-top:30px}.BuyBoxSection-683559780 .subscribe-buybox-nature-plus{background-color:#f3f3f3;flex-shrink:1;flex-grow:4;flex-basis:100%;background-clip:content-box;padding:0 15px;margin-top:30px}.BuyBoxSection-683559780 .title-readcube{display:block;margin:0;margin-right:20%;margin-left:20%;font-size:24px;line-height:32px;color:#222;padding-top:30px;text-align:center;font-family:Harding,Palatino,serif}.BuyBoxSection-683559780 .title-buybox{display:block;margin:0;margin-right:29%;margin-left:29%;font-size:24px;line-height:32px;color:#222;padding-top:30px;text-align:center;font-family:Harding,Palatino,serif}.BuyBoxSection-683559780 .title-asia-buybox{display:block;margin:0;margin-right:5%;margin-left:5%;font-size:24px;line-height:32px;color:#222;padding-top:30px;text-align:center;font-family:Harding,Palatino,serif}.BuyBoxSection-683559780 .asia-link{color:#069;cursor:pointer;text-decoration:none;font-size:1.05em;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;line-height:1.05em6}.BuyBoxSection-683559780 .access-readcube{display:block;margin:0;margin-right:10%;margin-left:10%;font-size:14px;color:#222;padding-top:10px;text-align:center;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;line-height:20px}.BuyBoxSection-683559780 .access-asia-buybox{display:block;margin:0;margin-right:5%;margin-left:5%;font-size:14px;color:#222;padding-top:10px;text-align:center;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;line-height:20px}.BuyBoxSection-683559780 .access-buybox{display:block;margin:0;margin-right:30%;margin-left:30%;font-size:14px;color:#222;opacity:.8px;padding-top:10px;text-align:center;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;line-height:20px}.BuyBoxSection-683559780 .usps-buybox{display:block;margin:0;margin-right:30%;margin-left:30%;font-size:14px;color:#222;opacity:.8px;text-align:center;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;line-height:20px}.BuyBoxSection-683559780 .price-buybox{display:block;font-size:30px;color:#222;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;padding-top:30px;text-align:center}.BuyBoxSection-683559780 .price-from{font-size:14px;padding-right:10px;color:#222;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;line-height:20px}.BuyBoxSection-683559780 .issue-buybox{display:block;font-size:13px;text-align:center;color:#222;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;line-height:19px}.BuyBoxSection-683559780 .no-price-buybox{display:block;font-size:13px;line-height:18px;text-align:center;padding-right:10%;padding-left:10%;padding-bottom:20px;padding-top:30px;color:#222;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif}.BuyBoxSection-683559780 .vat-buybox{display:block;margin-top:5px;margin-right:20%;margin-left:20%;font-size:11px;color:#222;padding-top:10px;padding-bottom:15px;text-align:center;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;line-height:17px}.BuyBoxSection-683559780 .button-container{display:flex;padding-right:20px;padding-left:20px;justify-content:center}.BuyBoxSection-683559780 .button-container >*{flex:1px}.BuyBoxSection-683559780 .button-container >a:hover,.Button-505204839:hover,.Button-1078489254:hover,.Button-2808614501:hover{text-decoration:none}.BuyBoxSection-683559780 .readcube-button{background:#fff;margin-top:30px}.BuyBoxSection-683559780 .button-asia{background:#069;border:1px solid #069;border-radius:0;cursor:pointer;display:block;padding:9px;outline:0;text-align:center;text-decoration:none;min-width:80px;margin-top:75px}.BuyBoxSection-683559780 .button-label-asia,.ButtonLabel-3869432492,.ButtonLabel-3296148077,.ButtonLabel-1566022830{display:block;color:#fff;font-size:17px;line-height:20px;font-family:-apple-system,BlinkMacSystemFont,”Segoe UI”,Roboto,Oxygen-Sans,Ubuntu,Cantarell,”Helvetica Neue”,sans-serif;text-align:center;text-decoration:none;cursor:pointer}.Button-505204839,.Button-1078489254,.Button-2808614501{background:#069;border:1px solid #069;border-radius:0;cursor:pointer;display:block;padding:9px;outline:0;text-align:center;text-decoration:none;min-width:80px;max-width:320px;margin-top:10px}.Button-505204839 .readcube-label,.Button-1078489254 .readcube-label,.Button-2808614501 .readcube-label{color:#069}
    /* style specs end */Subscribe to Nature+Get immediate online access to Nature and 55 other Nature journal$29.99monthlySubscribe to JournalGet full journal access for 1 year$199.00only $3.90 per issueAll prices are NET prices.VAT will be added later in the checkout.Tax calculation will be finalised during checkout.Buy articleGet time limited or full article access on ReadCube.$32.00All prices are NET prices.

    Additional access options:

    doi: https://doi.org/10.1038/d41586-022-03776-4

    References

    Subjects

    Conservation biology More

  • in

    Ecologists should create space for a wide range of expertise

    Madhusudan Katti says ecology would benefit from including perspectives from all of Earth’s inhabitants.Credit: Marc Hall

    Decolonizing science

    Science is steeped in injustice and exploitation. Scientific insights from marginalized people have been erased, natural history specimens have been taken without consent and genetics data have been manipulated to back eugenics movements. Without acknowledgement and redress of this legacy, many people from minority ethnic groups have little trust in science and certainly don’t feel welcome in academia — an ongoing barrier to the levels of diversity that many universities claim to pursue.
    In the next of a short series of articles about decolonizing the biosciences, Madhusudan Katti suggests five shifts that ecologists need to make to unravel the effects of colonization on their field. Katti, an evolutionary ecologist at North Carolina State University in Raleigh, would also like to see stronger inclusion of uncredentialed experts and Indigenous communities in research.

    Last year, my colleagues and I wrote a paper highlighting five shifts that would help to decolonize ecology (C. H. Trisos et al. Nature Ecol. Evol. 5, 1205–1212; 2021). Ecologists need to improve how they incorporate varied perspectives, approaches and interpretations from the diverse peoples inhabiting Earth’s natural environments. The five shifts are: the individual need to decolonize one’s mind; understand the history of colonization and how it shaped Western ecology; facilitate access to and dissemination of data; recognize diverse scientific expertise; and establish inclusive research groups. Although it can be difficult to make reforms given how resistant institutions are to change, we are optimistic because we have received invitations to speak on these issues. People are ready for these conversations.
    Decolonizing science toolkit
    My colleagues and I developed a workshop around the five shifts. We have conducted the workshop at my institution, and at the annual conference of the Society for Integrative and Comparative Biology. For each of the shifts, I have participants brainstorm and write down challenges and solutions that might lead to progress in these areas for their own research departments or institutions. We address them, shuffle groups and suggest policy changes and future action.Some organizations are already moving forward with some low-hanging fruit, such as making data and published results more accessible. However, open-access publishing models put an even greater burden of publication costs on authors and perpetuate inequalities, because early-career researchers and those in the global south often can’t afford them.The most contentious area tends to be the reluctance of academia to accept non-credentialed expertise such as traditional knowledge. Universities are in the business of giving out credentials in the form of degrees. If academia no longer requires a PhD, that can be a challenge to that model. There are also few, if any, incentives or rewards to spend time working towards decolonizing academia, even though it takes time and effort away from furthering individual careers.As an Indian American, I would like to see institutions expand antiracism conversations rather than introduce new checklists of things to do. For example, at annual meetings, it would be great to see scientific societies make more connections with the Indigenous communities where we work and invite them to share their perspectives.
    This interview has been edited for length and clarity. More

  • in

    Extinction magnitude of animals in the near future

    Selection of environmental-biotic events to be studiedIn global warming events associated with mass extinctions, the current environmental changes are similar to those recorded during the end-Ordovician, end-Guadalupian, and end-Permian mass extinctions. Therefore, I analyzed global surface temperature anomalies, mercury pollution concentrations, and deforestation percentages in these three mass extinctions and in the current crisis. The asteroid impact at the K–Pg boundary and nuclear war cause the formation of stratospheric soot aerosols distributed globally, thus inducing sunlight reductions and global cooling (impact winter and nuclear winter). I also analyzed stratospheric soot aerosols as a possible cause of future extinctions.Most likely case and worst caseThe most likely case corresponds to the reduction of CO2 emissions resulting from human conduct, the protection of forests, and the introduction of anti-pollution measures in the future under the Paris Agreement on Climate change and Sustainable Development Goals (SDGs). The worst case corresponds to the scenario in which humans fail to stop increasing global surface temperatures, pollution, and deforestation until 2100–2200 CE.I use the average of the RCP4.5 and RCP6.0 cases in the Intergovernmental Panel on Climate Change (IPCC)8 as the most likely case of GHG emissions, representing the middle of the four potential GHG emissions cases (RCP2.6, 4.5, 6.0, and 8.5) in Fifth Assessment Report of the IPCC8, approximately corresponding to the middle of SSP2-4.5 and SSP3-7.0 in Sixth Assessment Report of the IPCC9. The timing of decreased global GHG emissions is 2060–2080 CE. Therefore, I use the average GHG emissions and global surface temperature anomalies of the RCP4.5 and RCP6.0 cases as the most likely values and those of the RCP8.5 case as the worst-case scenario, marked by stopping GHG emissions from 2090 to 2100 CE8,9, as this case corresponds to the highest GHG emissions8,9.Surface temperature anomaly, environment, and extinction magnitude dataData on surface temperature anomalies and extinction percentages are from Kaiho4. Changes in industrial GHG emissions and global surface temperature anomalies are sourced from the Fifth and Sixth Assessment Report of the IPCC8,9.Pollution can be represented by mercury concentrations measured in sedimentary rocks recording mass extinctions8 and in recent sediments deposited in seas and lakes25,26 because mercury is toxic to plants and animals and because its sources include volcanic eruptions, meteorite impacts, and the combustion of fossil fuels10,33, which are common sources of pollutants, and because it can be commonly measured from sedimentary rocks recording mass extinctions33. The mercury concentration is related to the CO2 emission amount during global warming because of the common sources of mercury and CO2 (volcanism and fossil fuel combustion influencing global warming). Thus, the future mercury concentrations are estimated based on the CO2 emission amounts estimated by the IPCC8,9. Since mercury and the other pollutants mainly come from oil, coal, and vegetation33, the amount of mercury released should change in parallel with industrial CO2 emissions because there is a good correlation between mercury and CO2 emissions11.Deforestation occurs by the expansion of agricultural areas and urban areas, which are strongly related to human populations13,28. Thus, future deforestation percentages are estimated based on estimated future population data27 (Supplementary Table S2). The severity of deforestation in each event is expressed by the occupancy % of the deforested area in the pre-event forest area in (i) the Permian–Triassic transition marked by the largest mass extinction based on plant fossil records24 and (ii) 2005–2015 CE as a representative of the Anthropocene epoch12,13,28 based on the actual forest area relative to the pre-agriculture phase before 4000 BP. Deforestation is related to the human population because agriculture and urbanization have caused deforestation13,28. I estimate the past and future deforestation percentage using human population data in the past and future21 based on the parallel growth of the human population and deforestation13,28.Amount of stratospheric soot was calculated using a method of Kaiho and Oshima34 (Supplementary Table S1). I obtained global surface temperature anomaly caused by stratospheric soot using Fig. 5 of Kaiho and Oshima34.I then use those data to estimate the future extinction magnitude based on the assumption that the Earth and contemporary life at the time of each crisis are more or less mutually comparable throughout time and to the present day.I estimate the magnitude of the species animal extinction crisis between 2000 and 2500 CE using Figs. 1, 2 and Supplementary Tables S1 and S2 in each cause under the most likely case and worst case under three nuclear war scenarios (zero, minor, and major; Fig. 2d)15 in the PETM and mass extinction cases, respectively (Supplementary Tables S3, S4; Fig. 3). Finally, I estimate the magnitude of current animal extinction crisis by the four causes as an average of the species extinction magnitude by the four causes in Fig. 3. I use two different contribution rates of temperature anomalies, pollution, deforestation, and stratospheric soot by nuclear wars, 1:0.2:0.1:1 for marine animals and 1:0.5:1:1 for terrestrial tetrapods (different contribution case considering lower influence of pollution and deforestation to marine animals rather than terrestrial animals) and 1:1:1:1 for marine animals and 1:1:1:1 for terrestrial tetrapods (equal contribution case considering high influence of pollution and deforestation to marine animals via rain and soil erosion) (Supplementary Tables S5–S9). These contribution rates are estimated as end-members to show ranges of animal species extinction magnitude (%). More

  • in

    Extensive range contraction predicted under climate warming for two endangered mountaintop frogs from the rainforests of subtropical Australia

    Beniston, M., Diaz, H. F. & Bradley, R. S. Climatic change at high elevation sites: An overview. Clim. Change 36, 233–251 (1997).Article 

    Google Scholar 
    Chape, S., Spalding, M. & Jenkins, M. The world’s protected areas: Status, values, and prospects in the twenty-first century. Bioscience 59(7), 623–624 (2009).
    Google Scholar 
    Körner, C. Mountain biodiversity, its causes and function. Ambio 33, 11–17 (2004).Article 

    Google Scholar 
    Körner, C. et al. A global inventory of mountains for bio-geographical applications. Alp. Bot. 127, 1–15 (2017).Article 

    Google Scholar 
    Forero-Medina, G., Joppa, L. & Pimm, S. L. Constraints to species’ elevational range shifts as climate changes. Conserv. Biol. 25, 163–171 (2011).Article 
    PubMed 

    Google Scholar 
    Urban, M. C., Tewksbury, J. J. & Sheldon, K. S. On a collision course: Competition and dispersal differences create no-analogue communities and cause extinctions during climate change. Proc. R. Soc. B 279, 2072–2080 (2012).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Freeman, B. G., Scholer, M. N., Ruiz-Gutierrez, V. & Fitzpatrick, J. W. Climate change causes upslope shifts and mountaintop extirpations in a tropical bird community. Proc. Natl. Acad. Sci. 115, 11982–11987 (2018).Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar 
    Chen, I. C., Hill, J. K., Ohlemüller, R., Roy, D. B. & Thomas, C. D. Rapid range shifts of species associated with high levels of climate warming. Science 333, 1024 (2011).Article 
    CAS 
    PubMed 
    ADS 

    Google Scholar 
    Lenoir, J. & Svenning, J. C. Climate-related range shifts: A global multidimensional synthesis and new research directions. Ecography 38, 15–28 (2015).Article 

    Google Scholar 
    Parmesan, C. & Yohe, G. A globally coherent fingerprint of climate change impacts across natural systems. Nature 421, 37–42 (2003).Article 
    CAS 
    PubMed 
    ADS 

    Google Scholar 
    Román-Palacios, C. & Wiens, J. J. Recent responses to climate change reveal the drivers of species extinction and survival. Proc. Natl. Acad. Sci. 117, 4211–4217 (2020).Article 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar 
    Wiens, J. J. Climate-related local extinctions are already widespread among plant and animal species. PLoS Biol. 14, e200114 (2016).Article 

    Google Scholar 
    Orians, G. H. & Milewski, A. V. Ecology of Australia: The effects of nutrient-poor soils and intense fires. Biol. Rev. 82, 393–423 (2007).Article 
    PubMed 

    Google Scholar 
    Laurance, W. F. et al. The 10 Australian ecosystems most vulnerable to tipping points. Biol. Cons. 144, 1472–1480 (2011).Article 

    Google Scholar 
    Rahbek, C. et al. Humboldt’s enigma: What causes global patterns of mountain biodiversity?. Science 365, 1108–1113 (2019).Article 
    CAS 
    PubMed 
    ADS 

    Google Scholar 
    Williams, S. E., Bolitho, E. E. & Fox, S. Climate change in Australian tropical rainforests: An impending environmental catastrophe. Proc. R. Soc. Lond. B 270, 1887–1892 (2003).Article 

    Google Scholar 
    Mahony, M.J. The amphibians. in Remnants of Gondwana: A Natural and Social History of the Gondwana Rainforests of Australia. (eds. Kitching, R.L., Braithwaite, R., & Cavanaugh, J.) (Surrey Beatty & Sons, 2010).Kooyman, R. M., Watson, J. & Wilf, P. Protect Australia’s gondwana rainforests. Science 367, 1083–1083 (2020).Article 
    PubMed 
    ADS 

    Google Scholar 
    Narsey, S. et al. (2020). Impact of climate change on cloud forests in the Gondwana Rainforests of Australia World Heritage Area. Earth Systems and Climate Change Hub Report.Newell, D. An update on frog declines from the forests of subtropical eastern Australia in Status of Conservation and Decline of Amphibians: Australia, New Zealand, and Pacific Islands (eds. Heatwole H. and Rowley J. L.) 29–37 (CSIRO, 2018).DAWE. Bushfire Impacts Vol. 2021 (Commonwealth Department of Agriculture Water and Environment, 2020).
    Google Scholar 
    Collins, L. et al. The 2019/2020 mega-fires exposed Australian ecosystems to an unprecedented extent of high-severity fire. Environ. Res. Lett. 16, 044029 (2021).Article 
    ADS 

    Google Scholar 
    Filkov, A. I., Ngo, T., Matthews, S., Telfer, S. & Penman, T. D. Impact of Australia’s catastrophic 2019/20 bushfire season on communities and environment: Retrospective analysis and current trends. J. Saf. Sci. Resil. 1, 44–56 (2020).
    Google Scholar 
    Blunden, J. & Arndt, D. S. State of the climate in 2019. Bull. Am. Meteor. Soc. 101, S1–S429 (2020).Article 

    Google Scholar 
    Zhongming, Z., Linong, L., Wangqiang, Z. & Wei, L. AR6 Climate Change 2021: The Physical Science Basis (Springer, 2021).
    Google Scholar 
    Laidlaw, M. J., McDonald, W. J. F., Hunter, R. J., Putland, D. A. & Kitching, R. L. The potential impacts of climate change on Australian subtropical rainforest. Aust. J. Bot. 59, 440–449 (2011).Article 

    Google Scholar 
    Blaustein, A. R. et al. Direct and indirect effects of climate change on amphibian populations. Diversity 2, 281–313 (2010).Article 

    Google Scholar 
    Li, Y., Cohen, J. M. & Rohr, J. R. Review and synthesis of the effects of climate change on amphibians. Integr. Zool. 8, 145–161 (2013).Article 
    PubMed 

    Google Scholar 
    Carey, C. & Alexander, M. A. Climate change and amphibian declines: Is there a link?. Divers. Distrib. 9, 111–121 (2003).Article 

    Google Scholar 
    Cohen, J. M., Civitello, D. J., Venesky, M. D., McMahon, T. A. & Rohr, J. R. An interaction between climate change and infectious disease drove widespread amphibian declines. Glob. Change Biol. 25, 927–937 (2019).Article 
    ADS 

    Google Scholar 
    Geyle, H. M. et al. Red hot frogs: Identifying the Australian frogs most at risk of extinction. Pac. Conserv. Biol. 28, 211–223 (2021).Article 

    Google Scholar 
    Gillespie, G. R. et al. Status and priority conservation actions for Australian frog species. Biol. Conserv. 247, 108543 (2020).Article 

    Google Scholar 
    Almeida, A. M. et al. Prediction scenarios of past, present, and future environmental suitability for the Mediterranean species Arbutus unedo L. Sci. Rep. 12, 1–15 (2022).Article 

    Google Scholar 
    Lima, V. P. et al. Climate change threatens native potential agroforestry plant species in Brazil. Sci. Rep. 12, 1–14 (2022).Article 
    ADS 

    Google Scholar 
    Tiwari, S. et al. Modelling the potential risk zone of Lantana camara invasion and response to climate change in eastern India. Ecol. Process. 11(1), 1–13 (2022).Article 

    Google Scholar 
    Elith, J. et al. A statistical explanation of MaxEnt for ecologists. Divers. Distrib. 17, 43–57 (2011).Article 

    Google Scholar 
    Phillips, S. J., Anderson, R. P. & Schapire, R. E. Maximum entropy modeling of species geographic distributions. Ecol. Model. 190, 231–259 (2006).Article 

    Google Scholar 
    Galante, P. J. et al. The challenge of modeling niches and distributions for data-poor species: a comprehensive approach to model complexity. Ecography 41, 726–736 (2018).Article 

    Google Scholar 
    Li, J. et al. Climate refugia of snow leopards in High Asia. Biol. Conserv. 203, 188–196 (2016).Article 

    Google Scholar 
    Searcy, C. A. & Shaffer, B. H. Do ecological niche models accurately identify climatic determinants of species ranges?. Am. Nat. 187, 423–435 (2016).Article 
    PubMed 

    Google Scholar 
    Melo-Merino, S. M., Reyes-Bonilla, H. & Lira-Noriega, A. Ecological niche models and species distribution models in marine environments: A literature review and spatial analysis of evidence. Ecol. Model. 415, 108857 (2020).Article 

    Google Scholar 
    Anstis, M. Tadpoles and Frogs of Australia (New Holland Publishers Pty Limited, 2017).
    Google Scholar 
    Knowles, R., Mahony, M., Armstrong, J. & Donnellan, S. Systematics of sphagnum frogs of the Genus Philoria (Anura: Myobatrachidae) in Eastern Australia, with the description of two new species. Rec. Aust. Mus. 56, 57–74 (2004).Article 

    Google Scholar 
    Mahony, M. J. et al. A new species of Philoria (Anura: Limnodynastidae) from the uplands of the Gondwana Rainforests world heritage area of eastern Australia. Zootaxa 5104, 209–241 (2022).Article 
    PubMed 

    Google Scholar 
    Bolitho, L. J., Rowley, J. J. L., Hines, H. B. & Newell, D. Occupancy modelling reveals a highly restricted and fragmented distribution in a threatened montane frog (Philoria kundagungan) in subtropical Australian rainforests. Aust. J. Zool. 67, 231–240 (2021).Article 

    Google Scholar 
    Heard, G. et al. Post-fire impact assessment for priority frogs: northern Philoria. (NESP Threatened Species Recovery Hub Project 8.1.3 report, Brisbane, 2021).Vanderwal, J. All Future Climate Layers for Australia: 1 km Resolution (James Cook University, 2012).
    Google Scholar 
    Torkkola, J. J., Chauvenet, A. L. M., Hines, H. & Oliver, P. M. Distributional modelling, megafires and data gaps highlight probable underestimation of climate change risk for two lizards from Australia’s montane rainforests. Austral Ecol. 47(2), 365–379 (2021).Article 

    Google Scholar 
    Hijmans, R. J., Cameron, S. E., Parra, J. L., Jones, P. G. & Jarvis, A. Very high resolution interpolated climate surfaces for global land areas. Int. J. Climatol. 25, 1965–1978 (2005).Article 

    Google Scholar 
    Geoscience, A. Digital Elevation Model (DEM) 25 Metre Grid of Australia derived from LiDAR. (Geoscience Australia, 2015).Thuiller, W., Georges, D., Engler, R. & Breiner, F. (2014). biomod2: Ensemble platform for species distribution modeling. R package version 3.1-64. http://CRANR-project.org/package=biomod2. Accessed Feb 2021.Feng, X., Park, D. S., Liang, Y., Pandey, R. & Papeş, M. Collinearity in ecological niche modeling: Confusions and challenges. Ecol. Evol. 9, 10365–10376 (2019).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Thuiller, W. BIOMOD: Optimising predictions of species distributions and projecting potential future shifts under global change. Glob. Change Biol. 9, 1353–1362 (2003).Article 
    ADS 

    Google Scholar 
    MacKenzie, D. I., Nichols, J. D., Hines, J. E., Knutson, M. G. & Franklin, A. B. Estimating site occupancy, colonisation, and local extinction when a species is detected imperfectly. Ecology 84, 2200–2207 (2003).Article 

    Google Scholar 
    Schwalm, C. R., Glendon, S. & Duffy, P. B. RCP8.5 tracks cumulative CO2 emissions. Proc. Natl. Acad. Sci. 117, 19656–19657 (2020).Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar 
    Trisos, C. H., Merow, C. & Pigot, A. L. The projected timing of abrupt ecological disruption from climate change. Nature 580, 496–501 (2020).Article 
    CAS 
    PubMed 
    ADS 

    Google Scholar 
    Campos-Cerqueira, M. & Mitchell Aide, T. Lowland extirpation of anuran populations on a tropical mountain. PeerJ 2017, 1–10 (2017).
    Google Scholar 
    Pounds, J. A., Fogden, M. P. L. & Campbell, J. H. Biological response to climate change on a tropical mountain. Nature 398, 611–615 (1999).Article 
    CAS 
    ADS 

    Google Scholar 
    Raxworthy, C. J. et al. Extinction vulnerability of tropical montane endemism from warming and upslope displacement: A preliminary appraisal for the highest massif in Madagascar. Glob. Change Biol. 14, 1703–1720 (2008).Article 
    ADS 

    Google Scholar 
    Fordham, D. A. et al. Extinction debt from climate change for frogs in the wet tropics. Biol. Lett. 12, 20160236 (2016).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Hoffmann, E. P., Williams, K., Hipsey, M. R. & Mitchell, N. J. Drying microclimates threaten persistence of natural and translocated populations of threatened frogs. Biodivers. Conserv. 30(1), 15–34 (2020).Article 

    Google Scholar 
    Scheele, B. C., Driscoll, D. A., Fischer, J. & Hunter, D. A. Decline of an endangered amphibian during an extreme climatic event. Ecosphere 3, 101 (2012).Article 

    Google Scholar 
    Legge, S. et al. Rapid assessment of the biodiversity impacts of the 2019–2020 Australian megafires to guide urgent management intervention and recovery and lessons for other regions. Divers. Distrib. 28, 571–591 (2022).Article 

    Google Scholar 
    Canadell, J. G. et al. Multi-decadal increase of forest burned area in Australia is linked to climate change. Nat. Commun. 12, 6921 (2021).Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar 
    Hisano, M., Searle, E. B. & Chen, H. Y. H. Biodiversity as a solution to mitigate climate change impacts on the functioning of forest ecosystems. Biol. Rev. 93, 439–456 (2018).Article 
    PubMed 

    Google Scholar 
    Holz, A., Wood, S. W., Veblen, T. T. & Bowman, D. M. J. S. Effects of high-severity fire drove the population collapse of the subalpine Tasmanian endemic conifer Athrotaxis cupressoides. Glob. Change Biol. 21, 445–458 (2015).Article 
    ADS 

    Google Scholar 
    Hutley, L. B., Doley, D., Yates, D. J. & Boonsaner, A. Water balance of an australian subtropical rainforest at altitude: The ecological and physiological significance of intercepted cloud and fog. Aust. J. Bot. 45, 311–329 (1997).Article 

    Google Scholar 
    Godfree, R. C. et al. Implications of the 2019–2020 megafires for the biogeography and conservation of Australian vegetation. Nat. Commun. 12, 1023 (2021).Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar 
    Hennessy, K. et al. Climate Change Impacts on Fire-Weather in South-East Australia (Commonwealth Scientific and Industrial Research Organisation, 2005).
    Google Scholar 
    Moriondo, M. et al. Potential impact of climate change on fire risk in the Mediterranean area. Clim. Res. 31, 85–95 (2006).Article 

    Google Scholar 
    Pitman, A. J., Narisma, G. T. & McAneney, J. The impact of climate change on the risk of forest and grassland fires in Australia. Clim. Change 84, 383–401 (2007).Article 
    ADS 

    Google Scholar 
    Caughley, G. Directions in conservation biology. J. Anim. Ecol. 63, 215–244 (1994).Article 

    Google Scholar 
    Scheele, B. C. et al. Conservation translocations for amphibian species threatened by chytrid fungus: A review, conceptual framework, and recommendations. Conserv. Sci. Pract. 3, e524 (2021).
    Google Scholar 
    Rudin-Bitterli, T. S., Evans, J. P. & Mitchell, N. J. Geographic variation in adult and embryonic desiccation tolerance in a terrestrial-breeding frog. Evolution 74, 1186–1199 (2020).Article 
    CAS 
    PubMed 

    Google Scholar 
    Ashcroft, M. B. Identifying refugia from climate change. J. Biogeogr. 37, 1407–1413 (2010).
    Google Scholar 
    Keppel, G. et al. Refugia: Identifying and understanding safe havens for biodiversity under climate change. Glob. Ecol. Biogeogr. 21, 393–404 (2012).Article 

    Google Scholar 
    Selwood, K. E. & Zimmer, H. C. Refuges for biodiversity conservation: A review of the evidence. Biol. Conserv. 245, 108502 (2020).Article 

    Google Scholar  More

  • in

    Speciated mechanism in Quaternary cervids (Cervus and Capreolus) on both sides of the Pyrenees: a multidisciplinary approach

    Petronio, C. Les cervidés endémiques des îles méditerranéennes. Quaternaire 3–4, 259–264 (1990).
    Google Scholar 
    Liouville, M. Variabilité du Cerf élaphe (Cervus elaphus LINNE 1758) au cours du pléistocène moyen et supérieur en Europe occidentale : Approches morphométrique, paléoécologique et cynégétique (Museum National d’Histoire Naturelle, Paris, 2007).
    Google Scholar 
    van der Made, J., Stefaniak, K. & Marciszak, A. The polish fossil record of the wolf canis and the deer alces, capreolus, megaloceros, dama and cervus in an evolutionary perspective. Quatern. Int. 326–327, 406–430 (2014).
    Google Scholar 
    Guadelli, J.-L. Contribution à l’étude des zoocénoses préhistoriques en Aquitaine (Würm ancien et interstade würmiem. Universite de Bordeaux, Talence, 1987).
    Google Scholar 
    Guadelli, J.-L. Les cerfs du würm ancien en Aquitaine. Paléo 8, 99–108 (1996).
    Google Scholar 
    Defleur, A. et al. Le niveau moustérien de la grotte de l’Adaouste (Jouques, Bouches-du-Rhône): Approche culturelle et paléoenvironnements. Bull. Mus. anthropol. préhist. Monaco 37, 11–48 (1994).
    Google Scholar 
    Tournepiche, J.-F. Les grands mammifères pléistocènes de Poitou-Charente. Paléo 8, 109–141 (1996).
    Google Scholar 
    Delagnes, A. et al. Le gisement Pléistocène moyen et supérieur d’artenac (Saint-Mary, Charente): Premier bilan interdisciplinaire. Bull. Soc. Prehist. Fr. 96, 469–496 (1999).
    Google Scholar 
    Valensi, P., Psathi, E. & Lacombat, F. Le cerf élaphe dans les sites du Paléolithique moyen du Sud-Est de la France et de la Ligurie. Intérêts biostratigraphique, environnemental et taphonomique. In Acts of the XIVth UISPP Congress, Session 3: Paleoecology, General Sessions and Posters, 2–8 september 2001 97–105 (BAR International Series, 2004).Steele, T. E. Variation in mortality profiles of red deer (Cervus elaphus) in middle palaeolithic assemblages from western Europe. Int. J. Osteoarchaeol. 14, 307–320 (2004).
    Google Scholar 
    Croitor, R. A new form of wapiti cervus canadensis Erxleben, 1777 (Cervidae, Mammalia) from the late pleistocene of France. Palaeoworld 29, 789–806 (2020).
    Google Scholar 
    Meiri, M. et al. Subspecies dynamics in space and time: A study of the red deer complex using ancient and modern DNA and morphology. J. Biogeogr. 45, 367–380 (2018).
    Google Scholar 
    Queirós, J. et al. Red deer in Iberia: Molecular ecological studies in a southern refugium and inferences on European postglacial colonization history. PLoS ONE 14, e0210282 (2019).PubMed 
    PubMed Central 

    Google Scholar 
    Carranza, J., Salinas, M., de Andrés, D. & Pérez-González, J. Iberian red deer: paraphyletic nature at mtDNA but nuclear markers support its genetic identity. Ecol. Evol. 6, 905–922 (2016).PubMed 
    PubMed Central 

    Google Scholar 
    Rey-Iglesia, A., Grandal-d’Anglade, A., Campos, P. F. & Hansen, A. J. Mitochondrial DNA of pre-last glacial maximum red deer from NW Spain suggests a more complex phylogeographical history for the species. Ecol. Evol. 7, 10690–10700 (2017).PubMed 
    PubMed Central 

    Google Scholar 
    Geist, V. Deer of the world: Their evolution, behaviour, and ecology (Stackpole Books, Pennsylvania, 1998).
    Google Scholar 
    Rivals, F. & Lister, A. M. Dietary flexibility and niche partitioning of large herbivores through the pleistocene of Britain. Quatern. Sci. Rev. 146, 116–133 (2016).ADS 

    Google Scholar 
    Berlioz, E. Ecologie alimentaire et paléoenvironnements des cervidés européens du Pleistocène inférieur: le message des texutures de micro-usure dentaire (University of Poitiers, Poitiers, 2017).
    Google Scholar 
    Saarinen, J., Eronen, J., Fortelius, M., Seppä, H. & Lister, A. M. Patterns of diet and body mass of large ungulates from the pleistocene of Western Europe, and their relation to vegetation. Palaeontol. Electron. 19.3.32A, 1–58 (2016).
    Google Scholar 
    Stefano, G. D., Pandolfi, L., Petronio, C. & Salari, L. The morphometry and the occurrence of cervus elaphus (Mammalia, Cervidae) from the late Pleistocene of the Italian peninsula. Riv. Ital. Paleontol. Stratigr. 121, 103–120 (2015).
    Google Scholar 
    Terada, C., Tatsuzawa, S. & Saitoh, T. Ecological correlates and determinants in the geographical variation of deer morphology. Oecologia 169, 981–994 (2012).PubMed 
    ADS 

    Google Scholar 
    Sommer, R. S., Fahlke, J. M., Schmölcke, U., Benecke, N. & Zachos, F. E. Quaternary history of the European roe deer capreolus capreolus. Mammal Rev. 39, 1–16 (2009).
    Google Scholar 
    Lorenzini, R. et al. European Roe Deer Capreolus capreolus (Linnaeus, 1758). In Handbook of the Mammals of Europe (eds Hackländer, F. & Zachos, F. E.) 1–32 (Springer, Cham, 2022).
    Google Scholar 
    Lorenzini, R., Garofalo, L., Qin, X., Voloshina, I. & Lovari, S. Global phylogeography of the genus capreolus (Artiodactyla: Cervidae), a palaearctic meso-mammal. Zool. J. Linn. Soc. 170, 209–221 (2014).
    Google Scholar 
    Tixier, H. & Duncan, P. Are European roe deer browsers? A review of variations in the composition of their diets. Rev. Ecol. 51, 3–17 (1996).
    Google Scholar 
    Merceron, G., Viriot, L. & Blondel, C. Tooth microwear pattern in roe deer (Capreolus capreolus L.) from Chizé (Western France) and relation to food composition. Small Rumin. Res. 53, 125–132 (2004).
    Google Scholar 
    Delibes, J. R. Ecología y comportamiento del corzo (Capreolus capreolus L. 1758) en la Sierra de Grazalema (Cádiz) (Universidad Complutense, Complutense, 1996).
    Google Scholar 
    Hewitt, G. M. The genetic legacy of the quaternary ice ages. Nature 405, 907–913 (2000).CAS 
    PubMed 
    ADS 

    Google Scholar 
    Stewart, J. R., Lister, A. M., Barnes, I. & Dalén, L. Refugia revisited: Individualistic responses of species in space and time. Proc. R. Soc. Lond. B. Biol. Sci. 277, 661–671 (2010).
    Google Scholar 
    Álvarez-Lao, D. J. & García, N. Geographical distribution of pleistocene cold-adapted large mammal faunas in the Iberian peninsula. Quatern. Int. 233, 159–170 (2011).
    Google Scholar 
    Lumley, H. de. Le Paléolithique inférieur et moyen du Midi méditerranéen dans son cadre géologique. Tome I. Ligurie—Provence. Gall. Préhist. 5, (1969).Texier, P.-J. L’industrie moustérienne de l’abri pié-lombard (Tourettes-sur-Loup, Alpes-Maritimes). Bull. Soc. Préhist. Fr. 71, 429–448 (1974).
    Google Scholar 
    Texier, P.-J. et al. L’abri pié lombard à tourrettes-sur-loup (Alpes-Maritimes): Anciennes fouilles (1971–1985), nouvelles données. Bull. Mus.Anthropol.e Préhistor. Monaco 51, 19–49 (2011).
    Google Scholar 
    Tomasso, A. Territoires, systèmes de mobilité et systèmes de production : La fin du Paléolithique supérieur dans l’arc liguro-provençal (University of Nice Sophia Antipolis Nice, and University of Pisa, 2014).
    Google Scholar 
    Pelletier, M., Desclaux, E., Brugal, J.-P. & Texier, P.-J. The exploitation of rabbits for food and pelts by last interglacial neandertals. Quatern. Sci. Rev. 224, 105972 (2019).
    Google Scholar 
    Valladas, H. et al. Datations par la thermoluminescence de gisements moustériens du sud de la France. L’Anthropologie 91, 211–226 (1987).
    Google Scholar 
    Yokoyama, Y. et al. ESR dating of stalagmites of the Caune de l’Arago, the Grotte du Lazaret, the Grotte du Vallonnet and the abri Pié Lombard : a comparison with the U-Th method. In Third Specialist Seminar on TL and ESR Dating (eds. Hackens, T., Mejdahl, V., Bowman, S. G. E., Wintle, A. G. & Aitken, M. J.) 381–389 (1983).Romero, A. J., Fernández-Lomana, J. C. D. & Brugal, J.-P. Aves de caza. Estudio tafonómico y zooarqueológico de los restos avianos de los niveles musterienses de pié lombard (Alpes-Maritimes, Francia). Munibe Antropol. Arkeol. 68, 73–84 (2017).
    Google Scholar 
    Lumley (de), M.-A. Les néandertaliens dans le midi méditerranéen. In La Préhistoire française vol. T. 1 (Editions du CNRS, 1976).Porraz, G. En marge du milieu alpin. Dynamiques de formation des ensembles lithiques et modes d’occupation des territoires au paléolithique moyen (Université de Provence, Marseille, 2005).
    Google Scholar 
    Porraz, G. Middle Paleolithic mobile toolkits in shor-tterm human occupations: Two case studies. Eur. Prehist. 6, 33–55 (2009).
    Google Scholar 
    Roussel, A., Gourichon, L., Valensi, P. & Brugal, J.-P. Homme, gibier et environnement au Paléolithique moyen. Regards sur la gestion territoriale de l’espace semi-montagnard du Midi de la France. In Biodiversités, environnements et sociétés depuis la Préhistoire : nouveaux marqueurs et approches intégrées 87–99 (Éditions APDCA, 2021).Renault-Miskovsky, J. & Texier, J. Intérêt de l’analyse pollinique détaillée dans les concrétions de grotte .Application à l’abri pié-lombard (Tourettes-sur-Loup, Alpes maritimes). Quaternaire 17, 129–134 (1980).
    Google Scholar 
    Rosell, J. et al. A resilient landscape at teixoneres cave (MIS 3; Moià, Barcelona, Spain): The Neanderthals as disrupting agent. Quatern. Int. 435, 195–210 (2017).
    Google Scholar 
    Rosell, J. et al. Mossegades i Levallois: les noves intervencionsa la cova de les teixoneres (Moià, Bages). Trib d’Arqueologia 29–43 (2008).Rosell, J. et al. Los ocupaciones en la Cova de les Teixoneres (Moià, Barcelona): relaciones espaciales y grado de competencia entre hienas, osos y neandertales durante el Pleistoceno Superior. In Actas de la 1a Reunión de Científicos sobre Cubiles de Hiena (y Otros Grandes Carnívoros) en los Yacimientos Arqueológicos de la Península Ibérica (392–402) (eds Arriaza, M. C. et al.) (Museo Arqueológico Regional, 2010).
    Google Scholar 
    Rosell, J. et al. A stop along the way: The role of Neanderthal groups at level III of teixoneres cave (Moià, Barcelona, Spain). Quaternaire 21, 139–154 (2010).
    Google Scholar 
    Rosell, J. et al. Cova del toll y cova de les Teixoneres (Moià, Barcelona). In Los cazadores recolectores del Pleistoceno y del Holoceno en Iberia y el estrecho de Gibraltar (eds. Sala, R., Carbonell, E., Bermudez de Castro, J. M. & Arsuaga, J. L.) 302–307 (2014).Zilio, L. et al. Examining Neanderthal and carnivore occupations of teixoneres cave (Moià, Barcelona, Spain) using archaeostratigraphic and intra-site spatial analysis. Sci. Rep. 11, 4339 (2021).CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar 
    Tissoux, H. et al. Datation par les séries de l’uranium des occupations moustériennes de la grotte de teixoneres (Moia, Province de Barcelone, Espagne). Quaternaire 17, 27–33 (2006).
    Google Scholar 
    Talamo, S. et al. The radiocarbon approach to Neanderthals in a carnivore den site: A well-defined chronology for teixoneres cave (Moià, Barcelona, Spain). Radiocarbon 58, 247–265 (2016).CAS 

    Google Scholar 
    Álvarez-Lao, D. J., Rivals, F., Sánchez-Hernández, C., Blasco, R. & Rosell, J. Ungulates from teixoneres cave (Moià, Barcelona, Spain): Presence of cold-adapted elements in NE Iberia during the MIS 3. Palaeogeogr. Palaeoclimatol. Palaeoecol. 466, 287–302 (2017).
    Google Scholar 
    Rufà, A., Blasco, R., Rivals, F. & Rosell, J. Leporids as a potential resource for predators (hominins, mammalian carnivores, raptors): An example of mixed contribution from level III of teixoneres cave (MIS 3, Barcelona, Spain). C.R. Palevol. 13, 665–680 (2014).
    Google Scholar 
    Rufà, A., Blasco, R., Rivals, F. & Rosell, J. Who eats whom? Taphonomic analysis of the avian record from the middle paleolithic site of teixoneres cave (Moià, Barcelona, Spain). Quatern. Int. 421, 103–115 (2016).
    Google Scholar 
    Sánchez-Hernández, C., Rivals, F., Blasco, R. & Rosell, J. Short, but repeated Neanderthal visits to teixoneres cave (MIS 3, Barcelona, Spain): A combined analysis of tooth microwear patterns and seasonality. J. Archaeol. Sci. 49, 317–325 (2014).
    Google Scholar 
    Sánchez-Hernández, C., Rivals, F., Blasco, R. & Rosell, J. Tale of two timescales: Combining tooth wear methods with different temporal resolutions to detect seasonality of Palaeolithic hominin occupational patterns. J. Archaeol. Sci. Rep. 6, 790–797 (2016).
    Google Scholar 
    Picin, A. et al. Neanderthal mobile toolkit in short-term occupations at teixoneres cave (Moia, Spain). J. Archaeol. Sci. Rep. 29, 102165 (2020).
    Google Scholar 
    Fernández-García, M. et al. New insights in Neanderthal palaeoecology using stable oxygen isotopes preserved in small mammals as palaeoclimatic tracers in teixoneres cave (Moià, northeastern Iberia). Archaeol. Anthropol. Sci. 14, 106 (2022).
    Google Scholar 
    Ochando, J. et al. Neanderthals in a highly diverse, mediterranean-Eurosiberian forest ecotone: The pleistocene pollen record of teixoneres cave, Northeastern Spain. Quatern. Sci. Rev. 241, 106429 (2020).
    Google Scholar 
    López-García, J. M. et al. A multidisciplinary approach to reconstructing the chronology and environment of Southwestern European Neanderthals: The contribution of teixoneres cave (Moià, Barcelona, Spain). Quatern. Sci. Rev. 43, 33–44 (2012).ADS 

    Google Scholar 
    Sánchez-Hernández, C. et al. Dietary traits of ungulates in northeastern Iberian Peninsula: Did these Neanderthal preys show adaptive behaviour to local habitats during the middle palaeolithic?. Quatern. Int. 557, 47–62 (2020).
    Google Scholar 
    Fortelius, M. & Solounias, N. Functional characterization of ungulate molars using the abrasion-attrition wear gradient: A new method for reconstructing paleodiets. Am. Mus. Novit. 3301, 1–36 (2000).
    Google Scholar 
    Rivals, F., Solounias, N. & Mihlbachler, M. C. Evidence for geographic variation in the diets of late pleistocene and early holocene bison in North America, and differences from the diets of recent bison. Quatern. Res. 68, 338–346 (2007).ADS 

    Google Scholar 
    King, T., Andrews, P. & Boz, B. Effect of taphonomic processes on dental microwear. Am. J. Phys. Anthropol. 108, 359–373 (1999).CAS 
    PubMed 

    Google Scholar 
    Uzunidis, A. et al. The impact of sediment abrasion on tooth microwear analysis: An experimental study. Archaeol. Anthropol. Sci. 13, 134 (2021).
    Google Scholar 
    Kaiser, T. M. & Solounias, N. Extending the tooth mesowear method to extinct and extant equids. Geodiversitas 25, 321–345 (2003).
    Google Scholar 
    Xafis, A., Nagel, D. & Bastl, K. Which tooth to sample? A methodological study of the utility of premolar/non-carnassial teeth in the microwear analysis of mammals. Palaeogeogr. Palaeoclimatol. Palaeoecol. 487, 229–240 (2017).
    Google Scholar 
    Meadow, R. H. Early animal domestication in South Asia a first report of the faunal remains from mehrgarh Pakistan. In South Asian Archaeology (ed. Härtel, H.) 143–179 (Dietrich Reimer, Berlin, 1979).
    Google Scholar 
    Meadow, R. H. The use of size index scaling techniques for research on archaeozoological collections from the Middle East. In Historici Animalium ex. Ossibus Festschrift Angela Von Den Driesch Zum 65 Geburtstag (eds Becker, C. et al.) 285–300 (Verlag Marie Leidorf, Rahden, 1999).
    Google Scholar 
    Simpson, G. G. Large pleistocene felines of North America. Pleistocene felines North Am. 1136, 1–28 (1941).
    Google Scholar 
    Valli, A. M. F. & Guérin, C. L. gisement pléistocène supérieur de la grotte de Jaurens à Nespouls, Corrèze, France: Les cervidae (Mammalia, Artiodactyla). Publ. mus. Conflu. 1, 41–81 (2000).
    Google Scholar 
    Janis, C. M. The correlation between diet and dental wear in herbivorous mammals and its relationship to the determination of diets of extinct species. In Evolutionary Paleobiology of Behavior and Coevolution (ed. Boucot, A. J.) 241–259 (Elsevier, Amsterdam, 1990).
    Google Scholar 
    Heintz, E. Les Cervidés villafranchiens de France et d’Espagne (Museum National d’Histoire Naturelle, Parise, 1970).
    Google Scholar 
    Magniez, P. Etude paléontologique des artiodactyles de la grotte Tournal (Bize-Minervois, Aude, France) étude taphonomique, archéozoologique et paléoécologique des grands Mammifères dans leur cadre biostratigraphique et paléoenvironnemental (Université de Perpignan, Perpignan, 2010).
    Google Scholar 
    Cucchi, T., Hulme-Beaman, A., Yuan, J. & Dobney, K. Early neolithic pig domestication at Jiahu, Henan Province, China: clues from molar shape analyses using geometric morphometric approaches. J. Archaeol. Sci. 38, 11–22 (2011).
    Google Scholar 
    Evin, A. et al. The long and winding road: Identifying pig domestication through molar size and shape. J. Archaeol. Sci. 40, 735–743 (2013).
    Google Scholar 
    Pelletier, M., Kotiaho, A., Niinimäki, S. & Salmi, A.-K. Identifying early stages of reindeer domestication in the archaeological record: A 3D morphological investigation on forelimb bones of modern populations from Fennoscandia. Archaeol. Anthropol. Sci. 12, 169 (2020).PubMed 
    PubMed Central 

    Google Scholar 
    Bignon, O., Baylac, M., Vigne, J.-D. & Eisenmann, V. Geometric morphometrics and the population diversity of late glacial horses in Western Europe (Equus caballus arcelini): Phylogeographic and anthropological implications. J. Archaeol. Sci. 32, 375–391 (2005).
    Google Scholar 
    Pelletier, M. Morphological diversity, evolution and biogeography of early pleistocene rabbits (Genus Oryctolagus). Palaeontology 64, 817–838 (2021).
    Google Scholar 
    Curran, S. C. Expanding ecomorphological methods: Geometric morphometric analysis of cervidae post-crania. J. Archaeol. Sci. 39, 1172–1182 (2012).
    Google Scholar 
    Curran, S. C. Exploring eucladoceros ecomorphology using geometric morphometrics. Anat. Rec. 298, 291–313 (2015).
    Google Scholar 
    Cucchi, T. et al. Taxonomic and phylogenetic signals in bovini cheek teeth: Towards new biosystematic markers to explore the history of wild and domestic cattle. J. Archaeol. Sci. 109, 104993 (2019).
    Google Scholar 
    Jeanjean, M. et al. Sorting the flock: Quantitative identification of sheep and goat from isolated third lower molars and mandibles through geometric morphometrics. J. Archaeol. Sci. 141, 105580 (2022).
    Google Scholar 
    Herrera, P. L. Différences entre les dents jugales deciduales du cerf elaphe (Cervus Elaphus L.) et du boeuf domestique (Bos Taurus L.). Rev. Paléobiol. 8, 77 (1989).
    Google Scholar 
    Pfeiffer, T. Die stellung von dama (Cervidae, Mammalia) im system plesiometacarpaler hirsche des pleistozäns. Phylogenetische reconstruktion-metrische analyse. Cour Forsch. Senckenberg. 211, 1–218 (1999).
    Google Scholar 
    Rohlf, F. J. TPSDig, version 2.17 (Stony Brook, NY: Department of Ecology and Evolution, State University of New York, 2013).Bookstein, F. L. Morphometric Tools for Landmark Data: Geometry and Biology (Cambridge University Press, Cambridge, 1992).MATH 

    Google Scholar 
    Schlager, S. Morpho: Calculations and visualizations related to geometric morphometrics. (2013).Bookstein, F. L. Size and shape spaces for landmark data in two dimensions. Stat. Sci. 1, 181–222 (1986).MATH 

    Google Scholar 
    Kaiser, T. M. & Schulz, E. Tooth wear gradients in zebras as an environmental proxy—a pilot study. Mitt. Hambg. Zool. Mus. Inst. 103, 187–210 (2006).
    Google Scholar 
    Louys, J., Ditchfield, P., Meloro, C., Elton, S. & Bishop, L. C. Stable isotopes provide independent support for the use of mesowear variables for inferring diets in African antelopes. Proc. R. Soc. B. Biol. Sci. 279, 4441–4446 (2012).CAS 

    Google Scholar 
    Schulz, E. & Kaiser, T. M. Historical distribution, habitat requirements and feeding ecology of the genus equus (Perissodactyla). Mammal Rev. 43, 111–123 (2013).
    Google Scholar 
    Ulbricht, A., Maul, L. C. & Schulz, E. Can mesowear analysis be applied to small mammals? A pilot-study on leporines and murines. Mamm. Biol. 80, 14–20 (2015).
    Google Scholar 
    Danowitz, M., Hou, S., Mihlbachler, M., Hastings, V. & Solounias, N. A combined-mesowear analysis of late miocene giraffids from North Chinese and Greek localities of the pikermian biome. Palaeogeogr. Palaeoclimatol. Palaeoecol. 449, 194–204 (2016).
    Google Scholar 
    Marom, N., Garfinkel, Y. & Bar-Oz, G. Times in between: A zooarchaeological analysis of ritual in Neolithic Sha’ar Hagolan. Quatern. Int. 464, 216–225 (2018).
    Google Scholar 
    Ackermans, N. L. et al. Mesowear represents a lifetime signal in sheep (Ovis aries) within a long-term feeding experiment. Palaeogeogr. Palaeoclimatol. Palaeoecol. 553, 109793 (2020).
    Google Scholar 
    Mihlbachler, M. C., Rivals, F., Solounias, N. & Semprebon, G. M. Dietary change and evolution of horses in North America. Science 331, 1178–1181 (2011).CAS 
    PubMed 
    ADS 

    Google Scholar 
    Rivals, F., Rindel, D. & Belardi, J. B. Dietary ecology of extant guanaco (Lama guanicoe) from Southern Patagonia: Seasonal leaf browsing and its archaeological implications. J. Archaeol. Sci. 40, 2971–2980 (2013).
    Google Scholar 
    Rivals, F., Uzunidis, A., Sanz, M. & Daura, J. Faunal dietary response to the heinrich event 4 in southwestern Europe. Palaeogeogr. Palaeoclimatol. Palaeoecol. 473, 123–130 (2017).
    Google Scholar 
    Uzunidis, A., Rivals, F. & Brugal, J.-P. Relation between morphology and dietary traits in horse jugal upper teeth during the middle pleistocene in Southern France. Quat. Rev. Assoc. franc. l’étude Quat. 28, 303–312 (2017).
    Google Scholar 
    Uzunidis, A. Dental wear analyses of middle pleistocene site of Lunel-Viel (Hérault, France): Did equus and bos live in a wetland?. Quatern. Int. 557, 39–46 (2020).
    Google Scholar 
    Solounias, N. & Semprebon, G. Advances in the reconstruction of ungulate ecomorphology with application to early fossil equids. Am. Mus. Novit. 3366, 49 (2002).
    Google Scholar 
    Semprebon, G., Godfrey, L. R., Solounias, N., Sutherland, M. R. & Jungers, W. L. Can low-magnification stereomicroscopy reveal diet?. J. Hum. Evol. 47, 115–144 (2004).PubMed 

    Google Scholar 
    Grine, F. E. Dental evidence for dietary differences in australopithecus and paranthropus: A quantitative analysis of permanent molar microwear. J. Hum. Evol. 15, 783–822 (1986).
    Google Scholar 
    Teaford, M. F. & Oyen, O. J. In vivo and in vitro turnover in dental microwear. Am. J. Phys. Anthropol. 80, 447–460 (1989).CAS 
    PubMed 

    Google Scholar 
    Winkler, D. E. et al. The turnover of dental microwear texture: Testing the” last supper” effect in small mammals in a controlled feeding experiment. Palaeogeogr. Palaeoclimatol. Palaeoecol. 557, 109930 (2020).
    Google Scholar 
    Walker, A., Hoeck, H. N. & Perez, L. Microwear of mammalian teeth as an indicator of diet. Science 201, 908–910 (1978).CAS 
    PubMed 
    ADS 

    Google Scholar 
    Janis, C. M. & Lister, A. M. The morphology of the lower fourth premolaras a taxonomic character in the ruminantia (Mammalia; Artiodactyla), and the systematic position of triceromeryx. J. Paleontol. 59, 405–410 (1985).
    Google Scholar 
    Croitor, R. Animal husbandry and hunting. Bone material use ineconomic activities. In Kravchenko, E. A. (eds.) From Bronze to Iron: Pale-oeconomy of the Habitants of the Inkerman Valley (According the Materialof Excavations in Uch-Bash and Saharnaya Golovka Settlements). 191–222 (Institute of Archaeology of National Academy of Sciences of Ukraine, 2016).Geist, V. & Bayer, M. Sexual dimorphism in the cervidae and its relation to habitat. J. Zool. 214, 45–53 (1988).
    Google Scholar 
    Fichant, R. Le cerf: Biologie, comportement, gestion (Gerfaut Editions, 2003).
    Google Scholar 
    Arellano-Moullé, A. Les cervidés des niveaux moustériens de la grotte du Prince (Grimaldi, Vintimille, Italie) Etude paléontologique. Bull. Mus. Anthropol. Préhist. Monaco 39, 53–58 (1997).
    Google Scholar 
    Brugal, J. .-P. . La. faune des grands mammifères de l’abri des Canalettes – matériel 1980–1986. In L’abri des Canalettes Un habitat moustérien sur les grands Causses Nant Aveyron, 89–137 (ed. Meignen, L.) (CNRS Éditions, Paris, 1993).
    Google Scholar 
    La Gerber, J. P. faune des grands mammifères du Würm ancien dans le sud-est de la France (Université de Provence, Marseille, 1973).
    Google Scholar 
    Alonso, D. A. Analisis paleobiologico de los ungulados del pleistoceno superior de la meseta norte (Universidad de Salamanca, Salamanca, 2015).
    Google Scholar 
    Sanchez, B. La fauna de mamíferos del pleistoceno superior del Abric Romani (Capellades, Barcelona). Adas de Paleontol. 331–347 (1989).Clot, A. Le chevreuil, Capreolus capreolus (L.) (Ceervidae, Artiodactyla) dans le pléistocène de Ge$$rde (H.-P.) et des pyrénées. Bull. Soc. Hist. Nat. Toulouse 125, 83–86 (1989).
    Google Scholar 
    Vanpé, C. Mating systems and sexual selection in ungulates. New insights from a territorial species with low sexual size dimorphism: the European roe deer (Capreolus capreolus). (Université Paul Sabatier, Toulouse III and Swedish University of Agricultural Sciences, 2007).Horcajada-Sánchez, F. & Barja, I. Local ecotypes of roe deer populations (Capreolus capreolus L.) in relation to morphometric features and fur colouration in the centre of the Iberian Peninsula. Pol. J Ecol. 64, 113–124 (2016).
    Google Scholar 
    Semprebon, G. M., Sise, P. J. & Coombs, M. C. Potential bark and fruit browsing as revealed by Stereomicrowear analysis of the peculiar clawed herbivores known as Chalicotheres (Perissodactyla, Chalicotherioidea). J. Mammal. Evol. 18, 33–55 (2011).
    Google Scholar 
    Rivals, F. et al. Palaeoecology of the mammoth steppe fauna from the late pleistocene of the North Sea and Alaska: Separating species preferences from geographic influence in paleoecological dental wear analysis. Palaeogeogr. Palaeoclimatol. Palaeoecol. 286, 42–54 (2010).
    Google Scholar 
    Rivals, F., Takatsuki, S., Albert, R. M. & Macià, L. Bamboo feeding and tooth wear of three sika deer (Cervus nippon) populations from northern Japan. J. Mammal. 95, 1043–1053 (2014).
    Google Scholar 
    Lister, A. M. Evolutionary and ecological origins of British deer. Proc. R. Soc. Edinb. Sect. B. Biol. Sci. 82, 205–229 (1984).
    Google Scholar 
    Coulson, T., Guinness, F., Pemberton, J. & Clutton-Brock, T. The demographic consequences of releasing a population of red deer from culling. Ecology 85, 411–422 (2004).
    Google Scholar 
    Nussey, D. H., Clutton-Brock, T. H., Elston, D. A., Albon, S. D. & Kruuk, L. E. B. Phenotypic plasticity in a maternal trait in red deer. J. Anim. Ecol. 74, 387–396 (2005).
    Google Scholar 
    Frevert, W. Rominten (BLV Bayerischer Landwirtschaftsverlag, 1977).
    Google Scholar 
    Clutton-Brock, T. H. & Albon, S. D. Winter mortality in red deer (Cervus elaphus). J. Zool. 198, 515–519 (1982).
    Google Scholar 
    Loison, A. & Langvatn, R. Short- and long-term effects of winter and spring weather on growth and survival of red deer in Norway. Oecologia 116, 489–500 (1998).PubMed 
    ADS 

    Google Scholar 
    Torres-Porras, J., Carranza, J. & Pérez-González, J. Combined effects of drought and density on body and antler size of male iberian red deer cervus elaphus hispanicus: Climate change implications. Wildl. Biol. 15, 213–221 (2009).
    Google Scholar 
    Bugalho, M. N., Milne, J. A. & Racey, P. A. The foraging ecology of red deer (Cervus elaphus) in a mediterranean environment: Is a larger body size advantageous?. J. Zool. 255, 285–289 (2001).
    Google Scholar 
    Köhler, M. Skeleton and habitat of recent and fossil ruminants (F. Pfeil, Germany, 1993).
    Google Scholar 
    Boessneck, J. Zur grosse des mitteleuropaischen Rehes Capreolus capreolus L. in alluvial-vorgeschichtlicher und friiher historischer zeit. Z. f. Siiugetierkunde 21, 121–131 (1958).
    Google Scholar 
    Jensen, P. Body size trends of roe deer (Capreolus capreolus) from danish mesolithic sites. J. Dan. Archaeol. 10, 51–58 (1991).
    Google Scholar 
    Braza, F., San José, C., Aragon, S. & Delibes, J. R. El corzo andaluz. (Junta de Andalucía, 1994).Fandos, P. Skull biometry of spanish roe deer (Capreolus capreolus). Folia Zool. 43, 11–20 (1994).
    Google Scholar 
    Costa, L. First data on the size of north-Iberian roe bucks (Capreolus capreolus). Mammalia 59, 447–451 (1995).
    Google Scholar 
    Klein, D. R. & Strandgaard, H. Factors affecting growth and body size of roe deer. J. Wildl. Manag. 36, 64–79 (1972).
    Google Scholar  More