More stories

  • in

    The gut microbiome variability of a butterflyfish increases on severely degraded Caribbean reefs

    Kiers, E. T., Palmer, T. M., Ives, A. R., Bruno, J. F. & Bronstein, J. L. Mutualisms in a changing world: an evolutionary perspective. Ecol. Lett. 13, 1459–1474 (2010).Article 

    Google Scholar 
    Idjadi, J. & Edmunds, P. Scleractinian corals as facilitators for other invertebrates on a Caribbean reef. Mar. Ecol. Prog. Ser. 319, 117–127 (2006).Article 

    Google Scholar 
    Norström, A., Nyström, M., Lokrantz, J. & Folke, C. Alternative states on coral reefs: beyond coral–macroalgal phase shifts. Mar. Ecol. Prog. Ser. 376, 295–306 (2009).Article 

    Google Scholar 
    Richardson, L. E., Graham, N. A. J., Pratchett, M. S., Eurich, J. G. & Hoey, A. S. Mass coral bleaching causes biotic homogenization of reef fish assemblages. Glob. Chang. Biol. 24, 3117–3129 (2018).PubMed 
    Article 

    Google Scholar 
    Wilson, S. K., Graham, N. A. J., Pratchett, M. S., Jones, G. P. & Polunin, N. V. C. Multiple disturbances and the global degradation of coral reefs: are reef fishes at risk or resilient? Glob. Chang. Biol. 12, 2220–2234 (2006).Article 

    Google Scholar 
    Apprill, A. The role of symbioses in the adaptation and stress responses of marine organisms. Ann. Rev. Mar. Sci. 12, 291–314 (2020).Alberdi, A., Aizpurua, O., Bohmann, K., Zepeda-Mendoza, M. L. & Gilbert, M. T. P. Do Vertebrate gut metagenomes confer rapid ecological adaptation? Trends Ecol. Evol. 31, 689–699 (2016).PubMed 
    Article 

    Google Scholar 
    Voolstra, C. R. & Ziegler, M. Adapting with microbial help: microbiome flexibility facilitates rapid responses to environmental change. BioEssays 42, e2000004 (2020).Webster, N. S. & Reusch, T. B. H. Microbial contributions to the persistence of coral reefs. ISME J. 11, 2167–2174 (2017).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Wilkins, L. G. E. et al. Host-associated microbiomes drive structure and function of marine ecosystems. PLoS Biol. 17, e3000533 (2019).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ley, R. E. et al. Evolution of mammals and their gut microbes. Science 320, 1647–1651 (2008).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ley, R. E., Lozupone, C. A., Hamady, M., Knight, R. & Gordon, J. I. Worlds within worlds: evolution of the vertebrate gut microbiota. Nat. Rev. Microbiol. 6, 776–788 (2008).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Egerton, S., Culloty, S., Whooley, J., Stanton, C. & Ross, R. P. The gut microbiota of marine fish. Front. Microbiol. 9, 873 (2018).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Llewellyn, M. S., Boutin, S., Hoseinifar, S. H. & Derome, N. Teleost microbiomes: the state of the art in their characterization, manipulation and importance in aquaculture and fisheries. Front. Microbiol. 5, 1–1 (2014).Article 

    Google Scholar 
    Tarnecki, A. M., Burgos, F. A., Ray, C. L. & Arias, C. R. Fish intestinal microbiome: diversity and symbiosis unravelled by metagenomics. J. Appl. Microbiol. 123, 2–17 (2017).CAS 
    PubMed 
    Article 

    Google Scholar 
    Wang, A. R., Ran, C., Ringø, E. & Zhou, Z. G. Progress in fish gastrointestinal microbiota research. Rev. Aquac. 10, 626–640 (2018).Article 

    Google Scholar 
    Legrand, T. P. R. A., Wynne, J. W., Weyrich, L. S. & Oxley, A. P. A. A microbial sea of possibilities: current knowledge and prospects for an improved understanding of the fish microbiome. Rev. Aquac. 12, 1101–1134 (2019).Rawls, J. F., Mahowald, M. A., Ley, R. E. & Gordon, J. I. Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection. Cell 127, 423–433 (2006).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Shade, A. & Handelsman, J. Beyond the Venn diagram: the hunt for a core microbiome. Environ. Microbiol. 14, 4–12 (2012).CAS 
    PubMed 
    Article 

    Google Scholar 
    Sullam, K. E. et al. Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis. Mol. Ecol. 21, 3363–3378 (2012).PubMed 
    Article 

    Google Scholar 
    Ainsworth, T. D. et al. The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts. ISME J. 9, 2261–2274 (2015).CAS 
    Article 

    Google Scholar 
    Hernandez-Agreda, A., Leggat, W., Bongaerts, P. & Ainsworth, T. D. The microbial signature provides insight into the mechanistic basis of coral success across reef habitats. MBio. 7, e00560–16 (2016).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Roeselers, G. et al. Evidence for a core gut microbiota in the zebrafish. ISME J. 5, 1595–1608 (2011).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Clements, K. D., Angert, E. R., Montgomery, W. L. & Choat, J. H. Intestinal microbiota in fishes: what’s known and what’s not. Mol. Ecol. 23, 1891–1898 (2014).PubMed 
    Article 

    Google Scholar 
    Jones, J. et al. The microbiome of the gastrointestinal tract of a range-shifting marine herbivorous fish. Front. Microbiol. 9, 2000 (2018).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Miyake, S., Ngugi, D. K. & Stingl, U. Diet strongly influences the gut microbiota of surgeonfishes. Mol. Ecol. 24, 656–672 (2015).PubMed 
    Article 

    Google Scholar 
    Ngugi, D. K. et al. Genomic diversification of giant enteric symbionts reflects host dietary lifestyles. Proc. Natl Acad. Sci. USA 114, E7592–E7601 (2017).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Degregori, S., Casey, J. M. & Barber, P. H. Nutrient pollution alters the gut microbiome of a territorial reef fish. Mar. Pollut. Bull. 169, 112522 (2021).CAS 
    PubMed 
    Article 

    Google Scholar 
    Gómez, G. D. & Balcázar, J. L. A review on the interactions between gut microbiota and innate immunity of fish. FEMS Immunol. Med. Microbiol. 52, 145–154 (2008).PubMed 
    Article 
    CAS 

    Google Scholar 
    Butt, R. L. & Volkoff, H. Gut microbiota and energy homeostasis in fish. Front. Endocrinol. 10, 9 (2019).Article 

    Google Scholar 
    Hughes, T. P. et al. Spatial and temporal patterns of mass bleaching of corals in the Anthropocene. Science 359, 80–83 (2018).CAS 
    PubMed 
    Article 

    Google Scholar 
    Bellwood, D. R. et al. Evolutionary history of the butterflyfishes (f: Chaetodontidae) and the rise of coral feeding fishes. J. Evol. Biol. 23, 335–349 (2010).CAS 
    PubMed 
    Article 

    Google Scholar 
    Berumen, M., S., M. & McCormick, M. Within-reef differences in diet and body condition of coral-feeding butterflyfishes (Chaetodontidae). Mar. Ecol. Prog. Ser. 287, 217–227 (2005).Article 

    Google Scholar 
    Pratchett, M. S. Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef. Mar. Biol. 148, 373–382 (2005).Article 

    Google Scholar 
    Nagelkerken, I., van der Velde, G., Wartenbergh, S. L. J., Nugues, M. M. & Pratchett, M. S. Cryptic dietary components reduce dietary overlap among sympatric butterflyfishes (Chaetodontidae). J. Fish. Biol. 75, 1123–1143 (2009).CAS 
    PubMed 
    Article 

    Google Scholar 
    Bouchon & Harmelin-Vivien Impact of coral degradation on a chaetodontid fish assemblage, Moorea, French Polynesia. Fifth Int. Coral Tahiti 5, 427–432 (1985).
    Google Scholar 
    Graham, N. A. J. Ecological versatility and the decline of coral feeding fishes following climate driven coral mortality. Mar. Biol. 153, 119–127 (2007).Article 

    Google Scholar 
    Pratchett, M. S., Wilson, S. K. & Baird, A. H. Declines in the abundance of Chaetodon butterflyfishes following extensive coral depletion. J. Fish. Biol. 69, 1269–1280 (2006).Article 

    Google Scholar 
    Birkeland & Neudecker. Foraging behavior of two Caribbean Chaetodontids: Chaetodon capistratus and C. aculeatus. Copeia 1981, 169–178 (1981).Gore, M. A. Factors affecting the feeding behavior of a coral reef fish, Chaetodon capistratus. Bull. Mar. Sci. 35, 211–220 (1984).
    Google Scholar 
    Liedke, A. M. R. et al. Resource partitioning by two syntopic sister species of butterflyfish (Chaetodontidae). J. Mar. Biol. Assoc. UK 98, 1767–1773 (2018).CAS 
    Article 

    Google Scholar 
    Altieri, A. H. et al. Tropical dead zones and mass mortalities on coral reefs. Proc. Natl Acad. Sci. USA 114, 3660–3665 (2017).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Zaneveld, J. R., McMinds, R. & Vega Thurber, R. Stress and stability: applying the Anna Karenina principle to animal microbiomes. Nat. Microbiol. 2, 17121 (2017).CAS 
    PubMed 
    Article 

    Google Scholar 
    Neave, M. J., Apprill, A., Ferrier-Pagès, C. & Voolstra, C. R. Diversity and function of prevalent symbiotic marine bacteria in the genus Endozoicomonas. Appl. Microbiol. Biotechnol. 100, 8315–8324 (2016).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ricaboni, D., Mailhe, M., Khelaifia, S., Raoult, D. & Million, M. Romboutsia timonensis, a new species isolated from human gut. N. Microbes N. Infect. 12, 6–7 (2016).CAS 
    Article 

    Google Scholar 
    Zhang, L. et al. Characterization of the microbial community structure in intestinal segments of yak (Bos grunniens). Anaerobe 61, 102115 (2020).Gerritsen, J. et al. A comparative and functional genomics analysis of the genus Romboutsia provides insight into adaptation to an intestinal lifestyle. Preprint at bioRxiv https://doi.org/10.1101/845511 (2019).Fernández-Cadena, J. C. et al. Detection of sentinel bacteria in mangrove sediments contaminated with heavy metals. Mar. Pollut. Bull. 150, 110701 (2020).Williams, B., Landay, A. & Presti, R. M. Microbiome alterations in HIV infection a review. Cell. Microbiol. 18, 645–651 (2016).CAS 
    PubMed 
    Article 

    Google Scholar 
    Ahmed, H. I., Herrera, M., Liew, Y. J. & Aranda, M. Long-term temperature stress in the Coral Model Aiptasia supports the ‘Anna Karenina principle’ for bacterial microbiomes. Front. Microbiol. 10, 975 (2019).Beatty, D. S. et al. Variable effects of local management on coral defenses against a thermally regulated bleaching pathogen. Sci. Adv. 5, eaay1048 (2019).Zaneveld, J. R. et al. Overfishing and nutrient pollution interact with temperature to disrupt coral reefs down to microbial scales. Nat. Commun. 7, 11833 (2016).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ma, Q. et al. Impact of microbiota on central nervous system and neurological diseases: the gut-brain axis. J. Neuroinflammation 16, 53 (2019).Pita, L., Rix, L., Slaby, B. M., Franke, A. & Hentschel, U. The sponge holobiont in a changing ocean: from microbes to ecosystems. Microbiome 6, 46 (2018).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Johnson, K. V. A. & Foster, K. R. Why does the microbiome affect behaviour? Nat. Rev. Microbiol. 16, 647–655 (2018).CAS 
    PubMed 
    Article 

    Google Scholar 
    Werbner, M. et al. Social-stress-responsive microbiota induces stimulation of self-reactive effector T helper cells. mSystems 4, e00292-18 (2019).Keith, S. A. et al. Synchronous behavioural shifts in reef fishes linked to mass coral bleaching. Nat. Clim. Chang. 8, 986–991 (2018).Article 

    Google Scholar 
    Thompson, C. A., Matthews, S., Hoey, A. S. & Pratchett, M. S. Changes in sociality of butterflyfishes linked to population declines and coral loss. Coral Reefs 38, 527–537 (2019).Article 

    Google Scholar 
    Almany, G. R. Differential effects of habitat complexity, predators and competitors on abundance of juvenile and adult coral reef fishes. Oecologia 141, 105–113 (2004).PubMed 
    Article 

    Google Scholar 
    Clinchy, M., Sheriff, M. J. & Zanette, L. Y. Predator-induced stress and the ecology of fear. Funct. Ecol. 27, 56–65 (2013).Article 

    Google Scholar 
    Bolnick, D. I., Svanbäck, R., Araújo, M. S. & Persson, L. Comparative support for the niche variation hypothesis that more generalized populations also are more heterogeneous. Proc. Natl Acad. Sci. USA 104, 10075–10079 (2007).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Svanbäck, R. & Bolnick, D. I. Intraspecific competition drives increased resource use diversity within a natural population. Proc. R. Soc. B Biol. Sci. 274, 839–844 (2007).Article 

    Google Scholar 
    Neudecker, S. Foraging patterns of Chaetodontid and Pomacanthis fishes at St. Croix (U.S. Virgin Islands). Proc. Fifth International Coral Reef Symposium. 415–414 (1985).Lasker, H. Prey preferences and browsing pressure of the butterflyfish Chaetodon capistratus on Caribbean gorgonians. Mar. Ecol. Prog. Ser. 21, 213–220 (1985).Article 

    Google Scholar 
    Cole, A. J., Pratchett, M. S. & Jones, G. P. Diversity and functional importance of coral-feeding fishes on tropical coral reefs. Fish Fish. 9, 286–307 (2008).Article 

    Google Scholar 
    Pratchett, M. S., Wilson, S. K., Berumen, M. L. & McCormick, M. I. Sublethal effects of coral bleaching on an obligate coral feeding butterflyfish. Coral Reefs 23, 352–356 (2004).Article 

    Google Scholar 
    Fishelson, L., Montgomery, W. L. & Myrberg, A. A. A unique symbiosis in the gut of tropical herbivorous surgeonfish (Acanthuridae: teleostei) from the red sea. Science 229, 49–51 (1985).Article 

    Google Scholar 
    Miyake, S., Ngugi, D. K. & Stingl, U. Phylogenetic diversity, distribution, and cophylogeny of giant bacteria (Epulopiscium) with their surgeonfish hosts in the Red Sea. Front. Microbiol. 7, 285 (2016).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Choat, J. H., Robbins, W. & Clements, K. The trophic status of herbivorous fishes on coral reefs II. Mar. Biol. 145, 445–454 (2004).Article 

    Google Scholar 
    Elifantz, H., Horn, G., Ayon, M., Cohen, Y. & Minz, D. Rhodobacteraceae are the key members of the microbial community of the initial biofilm formed in Eastern Mediterranean coastal seawater. FEMS Microbiol. Ecol. 85, 348–357 (2013).CAS 
    PubMed 
    Article 

    Google Scholar 
    Pujalte, M. J., Lucena, T., Ruvira, M. A., Arahal, D. R. & Macián, M. C. In The Prokaryotes: Alphaproteobacteria and Betaproteobacteria (Springer, 2014).Glasl, B., Herndl, G. J. & Frade, P. R. The microbiome of coral surface mucus has a key role in mediating holobiont health and survival upon disturbance. ISME J. 10, 2280–2292 (2016).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Sunagawa, S. et al. Bacterial diversity and White Plague Disease-associated community changes in the Caribbean coral Montastraea faveolata. ISME J. 3, 512–521 (2009).CAS 
    PubMed 
    Article 

    Google Scholar 
    Roder, C. et al. Bacterial profiling of White Plague Disease in a comparative coral species framework. ISME J. 8, 31–39 (2014).CAS 
    PubMed 
    Article 

    Google Scholar 
    Morrow, K. M., Moss, A. G., Chadwick, N. E. & Liles, M. R. Bacterial associates of two caribbean coral species reveal species-specific distribution and geographic variability. Appl. Environ. Microbiol. 78, 6438–6449 (2012).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Chiarello, M. et al. Exceptional but vulnerable microbial diversity in coral reef animal surface microbiomes. Proc. R. Soc. B Biol. Sci. 287, 20200642 (2020).Article 

    Google Scholar 
    Sunagawa, S., Woodley, C. M. & Medina, M. Threatened corals provide underexplored microbial habitats. PLoS ONE 5, e9554 (2010).Zhang, C. et al. Ecological robustness of the gut microbiota in response to ingestion of transient food-borne microbes. ISME J. 10, 2235–2245 (2016).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Uren Webster, T. M. et al. Environmental plasticity and colonisation history in the Atlantic salmon microbiome: a translocation experiment. Mol. Ecol. 29, 886–898 (2020).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Fietz, K. et al. Mind the gut: genomic insights to population divergence and gut microbial composition of two marine keystone species. Microbiome 6, 82 (2018).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Smith, C. C., Snowberg, L. K., Caporaso, J. G., Knight, R. & Bolnick, D. I. Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota. ISME J. 9, 2515 (2015).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Uren Webster, T. M., Consuegra, S., Hitchings, M. & Garcia de Leaniz, C. Interpopulation variation in the Atlantic salmon microbiome reflects environmental and genetic diversity. Appl. Environ. Microbiol. 84, e00691-18 (2018).Fiore, C. L., Labrie, M., Jarett, J. K. & Lesser, M. P. Transcriptional activity of the giant barrel sponge, Xestospongia muta holobiont: molecular evidence for metabolic interchange. Front. Microbiol. 6, 364 (2015).Neave, M. J., Michell, C. T., Apprill, A. & Voolstra, C. R. Endozoicomonas genomes reveal functional adaptation and plasticity in bacterial strains symbiotically associated with diverse marine hosts. Sci. Rep. 7, 40579 (2017).Pogoreutz, C. et al. Dominance of Endozoicomonas bacteria throughout coral bleaching and mortality suggests structural inflexibility of the Pocillopora verrucosa microbiome. Ecol. Evol. 8, 2240–2252 (2018).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Reverter, M., Sasal, P., Tapissier-Bontemps, N., Lecchini, D. & Suzuki, M. Characterisation of the gill mucosal bacterial communities of four butterflyfish species: a reservoir of bacterial diversity in coral reef ecosystems. FEMS Microbiol. Ecol. 93 (2017).Parris, D. J., Brooker, R. M., Morgan, M. A., Dixson, D. L. & Stewart, F. J. Whole gut microbiome composition of damselfish and cardinalfish before and after reef settlement. PeerJ 4, e2412 (2016).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Reese, E. S. Coevolution of corals and coral feeding fishes of the family Chaetodontidae. In Proc. 3rd International Coral Reef Symposium, 267–274 (Rosenstiel School of Marine and Atmospheric Science, Miami, Florida., 1977).Hammer, T. J. & Bowers, M. D. Gut microbes may facilitate insect herbivory of chemically defended plants. Oecologia 179, 1–14 (2015).Kohl, K. D., Weiss, R. B., Cox, J., Dale, C. & Denise Dearing, M. Gut microbes of mammalian herbivores facilitate intake of plant toxins. Ecol. Lett. 17, 1238–1246 (2014).PubMed 
    Article 

    Google Scholar 
    Emslie, M. J., Pratchett, M. S., Cheal, A. J. & Osborne, K. Great Barrier Reef butterflyfish community structure: the role of shelf position and benthic community type. Coral Reefs 29, 705–715 (2010).Article 

    Google Scholar 
    Noble, M. M., Pratchett, M. S., Coker, D. J., Cvitanovic, C. & Fulton, C. J. Foraging in corallivorous butterflyfish varies with wave exposure. Coral Reefs 33, 351–361 (2014).Article 

    Google Scholar 
    Greb, L. et al. Ökologie und Sedimentologie eines rezenten Rampensystems an der Karibikküste von Panamá (Inst. für Geologie und Paläontologie, Stuttgart, 1996).Aronson, R., Hilbun, N., Bianchi, T., Filley, T. & McKee, B. Land use, water quality, and the history of coral assemblages at Bocas del Toro, Panamá. Mar. Ecol. Prog. Ser. 504, 159–170 (2014).Article 

    Google Scholar 
    Collin, R., D’Croz, L., Gondola, P. & Del Rosario, J. B. Climate and hydrological factors affecting variation in chlorophyll concentration and water clarity in the Bahia Almirante, Panama. Smithson. Contrib. Mar. Sci. 323–334 (2009).D’Croz, L., Rosario, J. B.del. & Gondola, P. The effect of fresh water runoff on the distribution of dissolved inorganic nutrients and plankton in the Bocas del Toro Archipelago, Caribbean Panamá. Caribb. J. Sci. 41, 414–429 (2005).
    Google Scholar 
    Seemann, J. et al. Assessing the ecological effects of human impacts on coral reefs in Bocas del Toro, Panama. Environ. Monit. Assess. 186, 1747–1763 (2014).CAS 
    PubMed 
    Article 

    Google Scholar 
    Guzmán, H. M., Barnes, P. A. G., Lovelock, C. E. & Feller, I. C. A site description of the CARICOMP mangrove, seagrass and coral reef sites in Bocas del Toro, Panamá. Caribb. J. Sci. 41, 430–440 (2005).
    Google Scholar 
    Beijbom, O. et al. Towards automated annotation of benthic survey images: variability of human experts and operational modes of automation. PLoS ONE 10, e0130312 (2015).PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 
    Rocha, L. A., Jogan, J., Király, G., Feráková, V. & Bernhardt, K.-G. Chaetodon capistratus. The IUCN Red List of Threatened Species. https://doi.org/10.2305/IUCN.UK.2010-4.RLTS.T165695A6094300.en (2010).Froese, R. & D. P. E. FishBase. FishBase. 2019. www.fishbase.org (2020)Smith, L. C. National Audubon Society Field Guide to Tropical Marine Fishes Caribbean, Gulf of Mexico, Florida, Bahamas, Bermuda (Alfred A. Knopf, 1997).Nguyen, B. N. et al. Environmental DNA survey captures patterns of fish and invertebrate diversity across a tropical seascape. Sci. Rep. 10, 1–14 (2020).Article 
    CAS 

    Google Scholar 
    Parada, A. E., Needham, D. M. & Fuhrman, J. A. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ. Microbiol. 18, 1403–1414 (2016).CAS 
    PubMed 
    Article 

    Google Scholar 
    Apprill, A., McNally, S., Parsons, R. & Weber, L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat. Microb. Ecol. 75, 129–137 (2015).Article 

    Google Scholar 
    Weber, L. et al. EMP 16S Illumina amplicon protocol. https://doi.org/10.17504/protocols.io.nuudeww (2018).R Core Team. R: a language and environment for statistical computing. (R Foundation for Statistical Computing, Vienna, Austria, 2019).
    Google Scholar 
    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10 (2011).Article 

    Google Scholar 
    Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581 (2016).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Wang, Q., Garrity, G. M., Tiedje, J. M. & Cole, J. R. Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 (2007).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596 (2013).CAS 
    PubMed 
    Article 

    Google Scholar 
    Wright, E. S. Using DECIPHER v2.0 to analyze big biological sequence data in R. R. J. 8, 352–359 (2016).Article 

    Google Scholar 
    Schliep, K., Potts, A. J., Morrison, D. A. & Grimm, G. W. Intertwining phylogenetic trees and networks. Methods Ecol. Evol. 8, 1212–1220 (2017).Article 

    Google Scholar 
    Weiss, S. et al. Normalization and microbial differential abundance strategies depend upon data characteristics. Microbiome 5, 27 (2017).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    McMurdie, P. J. & Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8, e61217 (2013).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Astudillo-García, C. et al. Evaluating the core microbiota in complex communities: a systematic investigation. Environ. Microbiol. 19, 1450–1462 (2017).PubMed 
    Article 

    Google Scholar 
    Dufrêne, M. & Legendre, P. Species assemblages and indicator species: the need for a flexible asymmetrical approach. Ecol. Monogr. 67, 345–366 (1997).
    Google Scholar 
    Roberts, D. W. labdsv: ordination and multivariate analysis for ecology. (2019).Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).CAS 
    PubMed 
    Article 

    Google Scholar 
    Leray, M. & Knowlton, N. Random sampling causes the low reproducibility of rare eukaryotic OTUs in Illumina COI metabarcoding. PeerJ 5, e3006 (2017).PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 
    Hill, M. O. Diversity and evenness: a unifying notation and its consequences. Ecology 54, 427–432 (1973).Article 

    Google Scholar 
    Alberdi, A. & Gilbert, M. T. P. A guide to the application of Hill numbers to DNA‐based diversity analyses. Mol. Ecol. Resour. 19, 1755–0998.13014 (2019).
    Google Scholar 
    Jost, L. Entropy and diversity. Oikos 113, 363–375 (2006).Article 

    Google Scholar 
    Chiu, C. H. & Chao, A. Estimating and comparing microbial diversity in the presence of sequencing errors. PeerJ 2016, e1634 (2016).Article 
    CAS 

    Google Scholar 
    Oksanen, J. et al. Community Ecology Package. Vienna R Found. Stat. Comput. https://doi.org/10.4135/9781412971874.n145 (2012).Chen, J. et al. Associating microbiome composition with environmental covariates using generalized UniFrac distances. Bioinformatics 28, 2106–2113 (2012).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Lozupone, C. A., Hamady, M., Kelley, S. T. & Knight, R. Quantitative and qualitative diversity measures lead to different insights into factors that structure microbial communities. Appl. Environ. Microbiol. 73, 1576–1585 (2007).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Jaccard, P. The distribution of the flora in the alpine zone.1. N. Phytol. 11, 37–50 (1912).Article 

    Google Scholar 
    Anderson, M. J., Ellingsen, K. E. & McArdle, B. H. Multivariate dispersion as a measure of beta diversity. Ecol. Lett. 9, 683–693 (2006).PubMed 
    Article 

    Google Scholar 
    Bray, J. R. & Curtis, J. T. An ordination of the upland forest communities of Southern Wisconsin. Ecol. Monogr. 27, 325–349 (1957).Article 

    Google Scholar 
    Anderson, M. J. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 26, 32–46 (2001).
    Google Scholar 
    Anderson, M. J. & Walsh, D. C. I. PERMANOVA, ANOSIM, and the Mantel test in the face of heterogeneous dispersions: what null hypothesis are you testing? Ecol. Monogr. 83, 557–574 (2013).Article 

    Google Scholar 
    Martinez Arbizu, P. pairwiseAdonis: pairwise multilevel comparison using adonis. R package version 0.3. https://github.com/pmartinezarbizu/pairwiseAdonis (2019).Roesch, L. F. W. et al. Pime: a package for discovery of novel differences among microbial communities. Mol. Ecol. Resour. 20, 415–428 (2020).CAS 
    PubMed 
    Article 

    Google Scholar 
    Breiman, L. Random forests. Mach. Learn. 45, 5–32 (2001).Article 

    Google Scholar 
    Klaus, J. S., Janse, I., Heikoop, J. M., Sanford, R. A. & Fouke, B. W. Coral microbial communities, zooxanthellae and mucus along gradients of seawater depth and coastal pollution. Environ. Microbiol. 9, 1291–1305 (2007).CAS 
    PubMed 
    Article 

    Google Scholar 
    Ward, R. J. et al. Gastrointestinal Bacterial Symbionts: Reproductive Strategy and Community Structure. Thesis, Cornell Univ. (2009).Séré, M. G. et al. Bacterial communities associated with Porites White Patch Syndrome (PWPS) on three Western Indian Ocean (WIO) coral reefs. PLoS ONE 8, e83746 (2013).PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 
    Moran, D., Turner, S. J. & Clements, K. D. Ontogenetic development of the gastrointestinal microbiota in the marine herbivorous fish Kyphosus sydneyanus. Microb. Ecol. 49, 590–597 (2005).CAS 
    PubMed 
    Article 

    Google Scholar 
    Mausz, M., Schmitz-Esser, S. & Steiner, G. Identification and comparative analysis of the endosymbionts of Loripes lacteus and Anodontia fragilis (Bivalvia: Lucinidae). (University of Vienna, 2008).Bano, N., DeRae Smith, A., Bennett, W., Vasquez, L. & Hollibaugh, J. T. Dominance of mycoplasma in the guts of the long-jawed mudsucker, Gillichthys mirabilis, from five California salt marshes. Environ. Microbiol. 9, 2636–2641 (2007).CAS 
    PubMed 
    Article 

    Google Scholar 
    Frade, P. R., Roll, K., Bergauer, K. & Herndl, G. J. Archaeal and Bacterial Communities associated with the surface mucus of Caribbean corals differ in their degree of host specificity and community turnover over reefs. PLoS ONE 11, e0144702 (2016).PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 
    Turnbaugh, P. J. et al. A core gut microbiome in obese and lean twins. Nature 457, 480–484 (2009).CAS 
    PubMed 
    Article 

    Google Scholar 
    Ley, R. E., Turnbaugh, P. J., Klein, S. & Gordon, J. I. Microbial ecology: human gut microbes associated with obesity. Nature 444, 1022–1023 (2006).CAS 
    PubMed 
    Article 

    Google Scholar 
    Kimes, N. E. et al. The Montastraea faveolata microbiome: ecological and temporal influences on a Caribbean reef-building coral in decline. Environ. Microbiol. 15, 2082–2094 (2013).PubMed 
    Article 

    Google Scholar 
    Smriga, S., Sandin, S. A. & Azam, F. Abundance, diversity, and activity of microbial assemblages associated with coral reef fish guts and feces. FEMS Microbiol. Ecol. 73, no–no (2010).Article 
    CAS 

    Google Scholar 
    Zhang, X. et al. Effects of dietary supplementation of Ulva pertusa and non-starch polysaccharide enzymes on gut microbiota of Siganus canaliculatus. J. Oceanol. Limnol. 36, 438–449 (2018).CAS 
    Article 

    Google Scholar 
    Klaus, J. S., Janse, I. & Fouke, B. W. Coral black band disease microbial communities and genotypic variability of the dominant cyanobacteria (CD1C11). Bull. Mar. Sci. 87, 795–821 (2011).Article 

    Google Scholar 
    Lu, J., Santo Domingo, J. W., Hill, S. & Edge, T. A. Microbial diversity and host-specific sequences of Canada goose feces. Appl. Environ. Microbiol. 75, 5919–5926 (2009).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ueki, A., Goto, K., Ohtaki, Y., Kaku, N. & Ueki, K. Description of Anaerotignum aminivorans gen. Nov., sp. nov., a strictly anaerobic, amino-acid-decomposing bacterium isolated from a methanogenic reactor, and reclassification of Clostridium propionicum, Clostridium neopropionicum and Clostridium lactatifermentans as species of the genus Anaerotignum. Int. J. Syst. Evol. Microbiol. 67, 4146–4153 (2017).CAS 
    PubMed 
    Article 

    Google Scholar 
    Bowman, K. S., Rainey, F. A. & Moe, W. M. Production of hydrogen by Clostridium species in the presence of chlorinated solvents. FEMS Microbiol. Lett. 290, 188–194 (2008).PubMed 
    Article 
    CAS 

    Google Scholar 
    Bueno de Mesquita, C. P., Sartwell, S. A., Schmidt, S. K. & Suding, K. N. Growing‐season length and soil microbes influence the performance of a generalist bunchgrass beyond its current range. Ecology 101, e03095 (2020).Clever, F. et al. The gut microbiome variability of a butterflyfish increases on severely degraded Caribbean reefs. Dryad Datasets. https://doi.org/10.5061/dryad.m905qfv28 (2022).Clever, F. & Scott, J. J. R code for reproducing the statistical analyses and figures of ‘The gut microbiome variability of a butterflyfish increases on severely degraded Caribbean reefs’. Commun. Biol. https://github.com/bocasbiome/web/ (2022). More

  • in

    Chlorophytes response to habitat complexity and human disturbance in the catchment of small and shallow aquatic systems

    Response of chlorophytes to environmental variables in field vs. forest pondsOur study demonstrated that human-originated transformation in the catchment area surrounding a small water body may influence the water conditions in terms of physical, chemical, and biological parameters as well as the ecological state of the aquatic environment in respect to green algae communities.Chlorophytes inhabiting field ponds were more abundant compared with the forest ponds. This shows that field ponds, due to the higher values of TRP and water conductivity, created favorable conditions for chlorophyte development. The high concentrations of TRP and conductivity in aquatic environments are characteristic in the case of agricultural catchments exposed to anthropogenic pressure because of the inflow from the surrounding fertilized fields42. In this type of pond, we also observed significantly higher water temperatures and pH due to the lack of trees around them compared to the forest ponds, two factors which also positively influenced the growth of chlorophytes. Both the higher light intensity and the smaller size of the field ponds cause earlier warming up than the forest ponds and give an advantage to high light tolerant species. Moreover, it is well known that an increase in temperature stimulates the release of phosphorus from the bottom sediments, so this could be another reason for the higher levels of TRP in the field ponds. Our CCA analysis showed that TRP and conductivity were the strongest determinants of the distribution of chlorophyte species in the examined water bodies. We found a large group of dominant species indicated high values of TRP (e.g. Ankistrodesmus falcatus, A. arcuatus, Monoraphidium griffithii, Pseudopediastrum boryanum, Pediastrum duplex, Scenedesmus obtusus, Scenedesmus arcuatus var. gracilis, Desmodesmus communis, Coelastrum microporum), and another group of species (e.g. Kirchneriella irregularis var. spiralis, Tetraedron minimum, Scenedesmus ecornis) that preferred high levels of conductivity.In the field ponds generally higher mean abundances of filtrators and Rotifera were observed. This could be another important factor stimulating the growth of chlorophytes and increasing their abundances by the resupply of nutrients through excretion43,44. On the other hand, the high densities of algae could be the factor that caused better zooplankton development, and therefore its abundance in field ponds was greater. Filtrating cladocerans and Rotifera also had a significant influence on the distribution of chlorophyte dominating species. However, even though the total abundance of both chlorophytes and filtering zooplankton was greater in the field ponds, CCA analysis revealed a negative relationship existing between filtrators and most dominant species of chlorophytes (e.g. Pandorina morum, Willea rectangularis, Desmodesmus armatus, Nephrochlamys willeana, Cosmarium trilobulatum). Only two chlorophyte species—Lemmermannnia tetrapedia and Tetraedron triangulare—co-occurred with cladoceran zooplankton. These latter species are very small compared to the species above and can therefore be overlooked by filtrators, which have a choice of larger and perhaps more nutritiously satisfying algae of the genus Pandorina, Crucigeniella, Cosmarium or Nephrochlamys, but still of a size suitable for zooplankton. It can also be interpreted in such a way that Crucigenia and Tetraedron are among the r-strategists that reproduce very quickly, so grazing pressure by zooplankton can stimulate their rapid development45 and thus they remain at a stable level.Specific environmental conditions prevailing in the field ponds resulted in a high number of exclusive taxa44, found only in this type of water body. Moreover, a greater diversity of the representatives of different functional groups were found here, compared to the forest ponds.Analyzing the distribution of chlorophytes in terms of phytoplankton functional groups39,40, we found that group W1 was represented by only one species, Gonium pectorale. This was especially noted in the field water bodies. This group is known to prefer small water bodies rich in organic matter from husbandry or sewage40, which suggests that the field catchment in our study migh be a supplier of these substances. It also proves that field surroundings are far more human impacted. In the field ponds we observed a higher abundance of chlorophytes belonging to the groups G (Eudorina elegans, Pandorina morum, Pandorina smithii and Volvox aureus), J (e.g. representatives of the genus Actinastrum, Chlorotetraedron, Coelastrum, Crucigenia, Desmodesmus/Scenedesmus, Golenkinia, Pediastrum, Tetraedron, Tetrastrum, Westella, Willea/Crucigeniella), W0 (genera Chlamydomonas, Chlorangiopsis, Chlamydomonadopsis, Planktococcomyxa/Coccomyxa) and X3 (Chlorella sp.), typical for shallow nutrient-rich waters (G and J), ponds with extremely high organic contents (W0), and for shallow well-mixed layers (X3), according to classification given by Padisak et al.40. Considering that nitrogen compounds had a similar level in both types of ponds it can be stated that the representatives of the above mentioned functional groups of chlorophytes associated with the field ponds were presumably dependent on higher concentrations of TRP and conductivity and not that much on nitrogen concentrations.In the forest ponds significantly higher values of water saturation were recorded compared to the field ponds. Moreover, the lack of inflow of fertilizers from the catchment area resulted in lower TRP concentrations, which along with lower water temperatures, pH and conductivity in the forest ponds may have contributed to the reduced abundance of chlorophytes compared to the field water bodies. RDA analysis showed that some dominant chlorophyte species (e.g. Closterium moniliferum, Closterium tumidulum, Cosmarium trilobulatum and Mougeotia sp.) were associated with this type of small water body. At the same time the abundance of these species was smaller in the field ponds. We also found that chlorophyte diversity (Shannon–Weaver index) was greater in the forest ponds. This suggests that water bodies located within the forested area, usually more natural ponds being less exposed to anthropogenic pressure, are characterized by greater biodiversity. Moreover, in this type of water body we found many exclusive species39, not reported from the field ponds. Interestingly, about the half of these taxa belonged to desmids, which prefer lower pH and conductivity46, conditions typical for forest ponds. This could be also a reason for the dominance of desmid species with the highest abundance/frequency, associated with forest ponds.Taking into consideration the phytoplankton functional groups39,40 our study showed that the chlorophytes associated with forest ponds prefer mesotrophic waters (from the group TD: Cladophora glomerata, Geminella turfosa, Geminella planctonica, Microspora sp., Netrium digitus, Oedogonium sp., Oocystidium ovale, Spirogyra sp. Zygnema sp. and those belonging to the group N: mainly genera Closterium, Cosmarium, Euastrum, Micrasterias, Staurastrum, Staurodesmus, Xanthidium). This explains their greater share in the less fertile forest ponds. Another group associated with the forest ponds – T (Mougeotia sp., Binuclearia lauterbornii) contains species tolerant to light deficiency, so they were able to develop well in the more shaded water bodies located in the forest catchment.Chlorophyte community structure in two types of habitats (open water vs. macrophyte-dominated zone)In our study, the type of habitat (open water and macrophyte-dominated zones) also had a significant structuring effect on chlorophytes. There were a group of species linked to the open water zone (Pandorina morum, Nephrochlamys willeana, Oocystis lacustris, Scenedesmus armatus, Scenedesmus intermedius and Desmodesmus communis), being negatively related to vegetated stations at the same time. Generally, we found here a higher mean abundance of chlorophytes compared to the macrophyte-dominated zones, possibly due to the higher values of nutrients such as NH4 and TRP, the conditions favouring the development of many algae species. The results of the CCA analysis with habitats confirmed the high importance of both nutritional factors in structuring the distribution of chlorophyte species. There was a group of species associated with a rise in the concentration of ammonium (e.g. Scenedesmus arcuatus var. gracilis, Pediastrum duplex, Closterium moniliferum, Closterium tumidulum, Cosmarium trilobulatum, Willea rectangularis) as well as with phosphates (Monoraphidium tortile, Scenedesmus ecornis, Tetradesmus lagerheimii and Tetraedron minimum). Generally, high abundance of chlorophytes in the open water area was accompanied by a small-sized fraction of zooplankton–rotifers. Therefore, rotifers had a lower impact on the distribution of chlorophytes than filtrators. The increasing numbers of cladocerans contributed to the lowering abundance of some chlorophytes, such as Monoraphidium tortile, Scenedesmus ecornis, Tetradesmus lagerheimii or Tetraedron minimum. This shows that filtrators, whose densities were significantly higher among macrophytes, were able to control the development of some chlorophyte species much more efficiently than small-bodied rotifers.The effect of habitat was also visible in the case of phytoplankton functional groups39,40. We found that representatives of the group N (e.g. Closterium, Cosmarium, Euastrum, Micrasterias, Staurastrum) had a significantly higher mean abundance in the open water zones compared to the macrophyte-dominated zones. Interestingly, according to Padisak et al.40 group N prefers less fertile (mesotrophic) conditions, which is inconsistent with our results. However, we think that their association with the open water sites could be connected rather with the place/level where they live in the water column, rather than with the trophic state of water. The above mentioned chlorophytes taxonomically belong to desmids, which are mostly benthic organisms. Their greater quantitative share in the samples from the open water areas could be an effect of the intensive water mixing in the shallow ponds due to the lack of macrophytes. Neustupa et al.47 confirm that desmids are able to form tychoplanktonic communities due to water movements. In the samples collected from the macrophyte-dominated stations the mean abundance of desmids was generally lower, probably because of the macrophyte stabilizing effect. Aquatic plants are known to reduce turbidity and stabilize bottom sediments48, so they can prevent any intensive water mixing in ponds. In the examined open water stations, we also found a higher mean abundance of chlorophytes typical for shallow nutrient-rich waters (group G: Eudorina, Pandorina, Volvox and group K: Radiococcus) and/or for ponds with extremely high organic contents (group W0: e.g. Chlamydomonas), which proves that the sites lacking macrophytes were more fertile. Additionally, clearly more representatives from the codon J and X1 (typical for waters with high trophic levels) and a greater diversity of the representatives of different functional groups were recorded in the open water area compared to the macrophyte-dominated zones.The macrophyte-dominated stations had more abundant communities of filtrators, as aquatic plants are known to provide a profitable shelter for zooplankton49. Cladoceran predominance among macrophytes may have been a force reducing green algae numbers. The chlorophytes of the investigated ponds were mostly small- or medium-size species. Their size distribution makes them a high quality food for zooplankton, particularly for cladoceran filtrators. According to RDA analysis apart from pond size, the presence of filtrators significanly reduced the abundance of several chlorophyte dominating species. The lower algae abundance among macrophytes compared to the open water zone could also be explained by competition between algae and macrophytes for light and nutrients37,50 and/or with the secretion of allelopathic substances e.g. by Ceratophyllum demersum51 inhibiting algal development. Our studies demonstrated that among chemical factors which clearly differentiated the two types of analysed habitat, TRP and NH4 significantly influenced the distribution of chlorophyte dominating species. The lower levels of these parameters in macrophyte-dominated zones suggest that the nutrient uptake by aquatic plants in the investigated water bodies was high. There are many reports on the decrease of nutrient concentrations by macrophytes30,37,52, which are consistent with our observations. Despite lower, compared to the open water zone, chlorophyte densities within the macrophyte-dominated zones there was a group of species (e.g. Mougeotia sp., Pediastrum tetras, Scenedesmus obtusus, Monoraphidium contortum) that selectively chose vegetated stands. Furthermore, we found a great number29 of exclusive chlorophyte species for macrophyte-dominated zones. Half of these taxa belong to desmids, which are often periphytic organisms associated with aquatic macrophytes53,54.Preference towards macrophyte-dominated stations was also documented for two phytoplankton functional groups (T: Mougeotia sp. and Binuclearia lauterbornii and TD: e.g., Spirogyra sp., Zygnema sp., Cladophora glomerata, Oedogonium sp.) and one group which occurred exlusively among vegetated sites (MP—Ulothrix). Interestingly, all the representatives of these groups had a similar filamentous morphological form, which suggests that many of them are of epithytic origin, coexisting within aquatic plants. Two more groups—X2 (Pseudodidymocystis/Didymocystis, Pteromonas) and W1 (Gonium pectorale) were clearly affected by the presence of macrophytes. According to Padisak et al.40, codons TD and X2 indicate mesoeutrophic conditions and their higher abundances in the macrophyte-dominated zones also proves that plants contribute to lowering the trophic levels in the examined ponds. On the other hand, the relatively high abundance of the representative of the group W1 in these habitats suggests that macrophytes could enrich ponds with organic matter during the process of their decomposition.Concluding, our results prove that different types of catchment area (field and forest) as well as different types of habitats (open water zone and macrophyte-dominated zone) create distinct, specific conditions (dependent on some physical–chemical and biological variables) for the occurrence of chlorophytes in small water bodies. We conclude that cosmopolitan chlorophytes undoubtedly respond to the level of habitat heterogeneity, contributing to the ecological assessment of small water bodies. Chlorophytes in particularl react to the level of human transformation in the ponds’ vicinities. This is why we suggest using them for water quality evaluation in ponds. This interdisciplinary research significantly broadens the knowledge, not only about the response of chlorophytes to physical–chemical parameters of water, but also about the food preferences of zooplankton for which green algae are the basic food, and vice versa about the impact of zooplankton on microalgae communities. The analyses provide valuable information on chlorophytes-zooplankton interactions and also about the relationships between chlorophytes and macrophytes. Received data emphasize the high value of field ponds, underestimated habitats particularly vulnerable to destruction in the agricultural landscape. The research will help to better understand the functioning of poorly studied small water bodies, which will contribute to the preservation of their biodiversity and protection against degradation. They will also be useful in the management of small water bodies based on the specificity of chlorophyte occurrence in various habitats and catchment type ponds. Moreover, these results are important in a broader context, as the interactions between the studied organisms and the physico-chemical parameters of water in small bodies of water are to some extent universal, so the analyses will broaden the knowledge about the functioning of larger bodies of water. More

  • in

    Estimation of nutrient loads with the use of mass-balance and modelling approaches on the Wełna River catchment example (central Poland)

    Case study areaThe studied catchment (2 621 km2) is located in the central-western part of Poland, and constitutes a part of the Oder River basin. The Wełna River (118 km) discharges to the Warta River near the town of Oborniki18, with an average flow rate of 8.1 m3s−1 (1980–2019) in this profile19. The natural conditions in this catchment favour the development of intensive agriculture, which covers almost 72% of this area (1888 km2) and contributes to the high consumption of mineral fertilizers20. Forest areas cover another 22% of this catchment (589 km2), while urbanised ones only 4% (93 km2) (Fig. 1). The Wełna River catchment is inhabited by approx. 230,000 people, of which only approx. 74% is served by wastewater treatment facilities21.Figure 1Localisation of the Wełna River catchment with its land use forms and nutrient sources. This figure was created using ArcGIS 10.2.1 for Desktop available at https://www.esri.com/en-us/home. Licence granted to Institute of Meteorology and Water Management.Full size imageInput dataBoth the mass-balance method and the modelling method require a similar amount and type of input data (Supplementary Table S1). Basic information on the Wełna River daily flow rates and nutrient concentrations in the closing profile of the catchment (Oborniki) has been obtained from the state monitoring services (Institute of Meteorology and Water Management—National Research Institute—IMGW-PIB13 and State Environmental Monitoring22—SEM) (Supplementary Table S1). They have formed the basis for the estimation of the share of individual sources in the mass-balance method, as well as for the calibration of the Macromodel DNS/SWAT in the modelling method. Other data, such as maps of elevation, river network and soil maps, as well as meteorological data, necessary for the development of an accurate representation of the studied catchment area on the Macromodel DNS/SWAT digital platform, were also obtained from state repositories. Data on the land use comes from the Corine Land Cover8, while detailed information on nutrient sources has been obtained mostly from the Local Data Bank of statistical information. The utilisation of the collected database has been presented in Fig. 2, and described in the following text. The comparison of the results for nutrient loads from both method was based on the year 2017, which was characterised by the maximum amount of monitoring data for both flows (365 measurements) (IMGW-PIB) and total nitrogen (TN) and total phosphorus (TP) (12 measurements–SEM). The average air temperature in 2017 in Poland was 1.5 °C higher than the long-term average (1971–2000) and was over 10 °C which resulted from the warm autumn and the end of the year. The time of the snow cover presence was shorter than the long-term data, and the rest of the year was classified as thermally normal.Figure 2Methodology diagram with relevant chapters marked in grey ovals (green—steps and data used for Mass Balance method, blue—steps data used for Modelling method, green/blue—steps and data used for both methods).Full size imageIn terms of precipitation, 2017 was assessed as wet, similarly due to rainy autumn and summer. In the Wełna River catchment area, the annual rainfall was about 770 mm, however high variability of precipitation conditions in particular months, from extremely wet to very dry, should be noted23. Therefore hydrologically, 2017 was considered normal with the flows only slightly lower than the long-term average.Mass-balance methodThe first method used for the quantification of sources and loads in the studied area was the static mass-balance method. It is widely used by the Polish administration authorities responsible for water management17. This method is based on the assumption that the sum of the nutrient loads in the river’s closing profile (selected based on access to the monitoring data) and its retention in the catchment equals the emission of nutrients in a given time. Such assumption allows the apportioning of the river loads among identified sources and the estimation of their contribution to the total loads, based on known or assumed values of their retention.River load calculationThe total load of nutrients discharged from the catchment was calculated using the daily flow rate and nutrient concentrations in the closing profile of the catchment area (Oborniki, Fig. 1) from the SEM (Supplementary Table S1). The daily river load was calculated using the following Eq. 5:$${L}_{river}=0.0864sum_{t=1}^{n=365}{({Q}_{t}cdot {C}_{t})}_{t}$$
    (1)
    where: Lriver is the annual load [kga-1], n is the number of days, t is the consecutive day, Ct is the concentration [mg L-1], Qt is the mean daily flow rate [Ls-1], and 0.0864 is the unit conversion.Due to the fact that the flow rate is measured daily and nutrient concentrations only 12 times a year, the linear interpolation method5 was used to obtain the daily concentration values:$${x}_{k}={x}_{a}+kcdot frac{{x}_{b}-{x}_{a}}{n+1}$$
    (2)
    where: xk is the interpolated concentration value, xa is the first of the two measured concentration values between which the concentrations are interpolated, xb is the second of the two measured concentration values between which the concentrations are interpolated, k is the consecutive number of missing value and n is number of missing values.Source apportionmentFor the mass-balance method, data on nutrient loads for source apportionment (emission inventory) was collected in order to proceed with further calculations. The calculations were performed for 2017, due to the availability of river monitoring data and the nutrient sources were divided into 7 categories, based on the HELCOM guidelines5: municipal (MWS) and industrial (INS) point sources, municipal diffuse sources (SCS), forestry (MFS), agriculture (AGS), natural background (NBS) and atmospheric deposition (ATS). The category of “unknown sources” (UKS) was taken into account, in order to include possible discrepancies in nutrients load apportionment, and to cover eventual differences between calculated river load and inventoried emission.The MWS loads were calculated on the basis of the number of inhabitants served by the wastewater treatment plants (WWTPs)21. In the Wełna River catchment, 151 771 inhabitants were served by the 12 WWTPs covered by the National Wastewater Treatment Program (NWTP)24, which provides information on the total discharge volume from each facility. For 5 of these plants, information on influent/effluent nutrient concentrations was also available, allowing the direct calculation of discharged loads. For the remaining seven facilities, the loads were calculated on the basis of the mean influent concentration information, available for the WWTPs covered by the NWTP (80 mgL−1 and 12 mgL−1 for TN and TP, respectively), and approximated nutrient reduction level in non-biological WWTPs. This reduction level, based on data from the NWTP, was set at 65% for TN and 35% for TP24. Another 19 350 inhabitants of this catchment were connected to the small WWTPs, not included in the NWTP. This part of the MWS load was calculated using the mean daily wastewater outflow (0.12 m3day−1 per person), the same mean nutrient concentrations and reduction levels as presented above. Additionally, the remaining 25% of the catchment’s population (58,000) is not connected to any WWTP and uses septic tanks and other types of individual wastewater treatment systems. The load from this source was expressed as SCS, and calculated using unit loads set on 11 gday−1 per person and 1.6 gday-1 per person for TN and TP, respectively17. The industrial nutrient input information (INS) was gathered directly from the Statistics Poland office database21.The AGS load was calculated using nitrate and phosphate concentrations in shallow groundwater (90 cm below the ground surface), from 22 sampling points located on agricultural areas in the Wełna River catchment17. Concentrations were recalculated to TN and TP respectively and averaged. Thus, the obtained mean concentrations were 8.25 mgL−1 of TN and 1.92 mgL−1 of TP. Subsequently, load values were calculated by multiplying these concentrations by the outflow from agricultural areas, calculated as a share of the total catchment outflow respective to the agricultural use of the area. The calculated loads were multiplied by coefficients reflecting the share of monitored outflow (groundwaters and tile drainage) from the agricultural runoff (1.11 for TN and 4.17 for TP)25. Subsequently, the natural background (NBS) was subtracted from the AGS load.The load corresponding to NBS was calculated using the total catchment outflow and nutrient concentration values reflecting conditions in undisturbed areas of pre-human activity, set as 0.15 mgL−1 and 0.02 mgL−1, for TN and TP respectively17. The MFS load was also calculated in a similar way, using nutrient concentrations set to represent forest catchment as 0.31 mgL−1 and 0.038 mgL−117 and the outflow calculated as the share of the total catchment area, respective for the catchment part covered by forest. Also in this case, the NBS load has been subtracted. As for the ATS load, data on pollutant deposition into the ground from precipitation was taken from the SEM network26. This data was based on precipitation and its chemistry measurements taken from 22 monitoring stations covering the entire territory of Poland. The total load from the point and diffuse sources was calculated by adding the loads mentioned above. The eventual difference between the river load (“River load calculation” Section) and inventoried emission (“Mass-balance method” Section) accounted for the other sources (UKS).Load apportionmentThe contribution of each source to the calculated river load was calculated based on a simplified equation modified from HELCOM5:$${L}_{river}=DP+LOD-RP-RD$$
    (3)
    where: Lriver is the river load [kga−1], DP is the load from point sources (MWS and INS) [kga−1], LOD is the load from diffuse sources (SCS, ATS, MFS, AGS and, NBS) [kga−1], RP is the point source retention [kga−1] and RD is the diffuse source retention [kga−1].In the adopted mass-balance method, it is assumed that nutrient load from the point sources (DP) is introduced directly into the river bed phase, while load from the diffuse sources (LOD) is discharged into both phases of the catchment, land and river bed ones. In both phases, self-purification processes are taking place, resulting in the reduction of nutrient loads on the way from the source to the catchment closing profile. However, due to the limited amount of data, the self-purification processes in the river have been omitted, therefore the point source retention (RP) equalled 0 kga−1. Subsequently, the diffuse source retention (RD) has been estimated on the basis of the difference between each nutrient load of the river (Lriver) and the point sources (LOD). The remaining river load has been then attributed proportionally to the contribution of the particular diffuse sources to the total source apportionment (emission inventory).Modelling methodThe digital platform, the Macromodel DNS with the SWAT module27,28,29,30,31,32, was used for comparison for the nutrient balancing method described in “Mass-balance method” Section. This advanced dynamic tool tracks nitrogen and phosphorus migration paths in the river basin taking into account their spatial and temporal variability. For this purpose, it takes into account a very extensive input database, similar to that used in the mass balance method (Supplementary Table S1). Natural and anthropogenic processes that affect the transport and transformation of nutrients, are also part of this platform. The SWAT module (version 2012) is a tool which operates in the spatial information system (GIS) and is fully integrated with it. Using the digital elevation model (DEM), the SWAT module divided the entire analysed Wełna River catchment into a total of 225 sub-catchments of an average area of 11.5 km2. The subsequent use of data on land use (forests, agriculture and urbanised areas) and the types of soils (31 classes) allowed the authors to identify a total of 2824 hydrological response units (HRUs), homogeneous in terms of vegetation, soil and topography33. Afterwards, a simulation of soil water content, evapotranspiration, surface runoff, primary and underground flows was carried out in accordance with the water balance Eq. (4), which represents the basis for the quantitative component and the HRU.$${SW}_{t}={SW}_{0}+sum_{i=1}^{t}({R}_{day}-{Q}_{surf}-{E}_{a}-{W}_{seep}-{Q}_{gw})$$
    (4)
    where: SWt is the final soil water content (mm H2O), SW0 is the initial soil water content (mm H2O), Rday is the amount of precipitation (mm H2O), Qsurf is amount of surface runoff (mm H2O), Ea is the amount of evapotranspiration (mm H2O), Wseep is the amount of water entering the vadose zone from the soil profile (mm H2O), Qgw is the amount of return flow (mm H2O).The model directs all runoff flows generated by each HRU through the channel network, thus simulating a catchment. The water balance equation also represents a basis for the simulation, transport and transformation of nutrients required for the quantitative component of the model. This tool models forms of nitrogen, organic and inorganic , different forms of phosphorus in soil34, as well as organic nitrogen and phosphorus forms associated with plant residues, microbial biomass and soil humus35,36,37,38. Final results of simulations are produced by the SWAT model as all the forms of nitrogen and phosphorus (in kilograms of N and P per a time unit, respectively) are then summed up to give TN and TP values. To verify that the model properly predicts TN and TP values its results are calibrated with the TN and TP values resulting from SEM, as described in Sect. 2.4.1. Moreover, the particular forms of nitrogen and phosphorus have also been compared with the modelling results (Supplementary Table S4). A detailed overview of the migration and transformation pathways of nitrogen and phosphorus forms in the catchment has been presented in the Supplementary Information (Sect. S1), while mathematical description of these processes is included in the theoretical documentation of the SWAT model39.Similarly, as in the case of the mass-balance method, diffuse sources of nutrients from agriculture (AGS), forestry (MFS) or urban areas (URB) in SWAT were simulated in the land phase of the catchment. In the land phase, the model simulates both the infiltration of nutrients into the soil (fertilization, plant biomass, precipitation) and their removal from it (volatilization, denitrification, erosion, surface runoff). Additionally, changes in the distribution of nutrients in the soil (uptake by plants) and the low mobility of phosphorus itself are also taken into account39,40,41.Pollutants from municipal and industrial point sources (MWS, INS) are introduced directly into the river bed phase. The exception here is the nutrient load from municipal diffuse sources (SCS) which, reduced as a result of the self-purification processes taking place in the land phase, is also treated in the model as point sources. The SCS nutrient load mainly derives from leaking or illegally emptied septic tanks. For this purpose, septic tanks have been divided into three types: leaky, partially illegally emptied, and sealed septic tanks, legally emptied. The loads from the legally emptied tanks are included in the effluents from WWTPs reported in the catchment. While for the remaining types, their loads are calculated using factors depending on their effectiveness in nutrient removal (15 – 50%). The final nutrient load derived from these types of facilities is then calculated, taking into consideration the number of inhabitants using the different types of septic tanks and the average chemical composition of wastewater21.The load of nutrients from the atmospheric deposition (ATS) affects both land and river phases due to the presence of two deposition mechanisms in the SWAT module, i.e., wet and dry deposition. The model also allows for the determination of nutrient loads generated as a result of natural processes of nitrogen and phosphorus transformation and transport in the soil, with the omission of all anthropogenic pressure—natural background (NBS).Calibration, verification and validationThe SWAT module for the Wełna River has been calibrated, verified and validated using the SWAT-CUP software42. For the quantitative component (water circulation in the catchment), the implemented daily flow data (source: IMGW-PIB) for the period of 18 years (2001–2018) came from the water gauge stations on the Wełna River (Pruśce and Kowanówko) and its tributary (the Flinta River-Ryczywół) (Fig. 1). The qualitative component (nitrogen and phosphorus concentration in the catchment) was gathered from the SEM stations localised at the Wełna River (Oborniki and Rogoźno) (Fig. 1) and covered a period of 13 years (2005–2018). Three statistical measures, coefficient of determination (R2)43, percent bias (PBIAS)44, and Kling-Gupta efficiency (KGE)45, have been used to indicate the Wełna River model performance (Supplementary Tables S2 and S3). In terms of the quantitative component, the calibration and verification coefficients R2, KGE and PBIAS classified the model performance generally as good and very good for the main river (Wełna), and satisfactory and good for its tributary (Flinta). During the validation procedure, all coefficient values rated the model performance for daily flow simulations as very good. In terms of qualitative components, the model performance for TN simulations can be considered as very good or good, according to the all-applied coefficients. Lower model performance, mostly satisfactory, was observed for TP mainly due to the variability of phosphorus temporal distribution patterns (Supplementary Table S2). The entire process was described in detail in Orlińska-Woźniak et al46.Variant scenariosIn order to determine the contribution of individual sources to the total load of nutrients in the profile closing the analysed catchment, a final simulation of the model was used and subjected to calibration, verification and validation procedures, and called the baseline scenario (A0). Subsequent so-called variant scenarios (A1–A5), i.e. model simulations, were developed. In variant scenarios the values of selected parameters were changed in relation to the A0 scenario. This was used both in the river bed phase for point sources (A1) and for individual diffuse sources (A2–5), thus imitating surface changes for particular types of land use in the land phase of the catchment (Fig. 3).Figure 3Variant analysis diagram for assessment of nutrient loads (L) for particular modelling scenarios and sources described in the text: MWS, INS, SCS—point sources, URB—urban, AGS—agricultural, MFS—forest.Full size imageIn the A1 scenario, all parameterized and aggregated point sources (MWS, INS, SCS) for each relevant sub-basin (LMWS,INS,SCS), were removed from the model to calculate their contribution to the total nutrient loads in the closing profile of the studied catchment (LA1).In the next two scenarios (A2 and A3), concerning urban and agricultural land use, their surface areas (5 663 ha and 192 917 ha, respectively) were successively replaced by the forest land use. This procedure was based on the assumption that the forest is the primary type of land use for this catchment area47. In order to completely eliminate the influence of these areas, the nutrient loads from the relevant surface area occupied by forest land use were subtracted, in order to estimate the contribution of urban and agricultural land (LURB and LAGS, respectively).The change in land use from urbanised and agricultural areas to forest areas increased their percentage of the catchment area to almost 100%, thus the original image of the catchment area and the nutrient load at its mouth. On this basis, in scenario A4, the nutrient load from forests LMFS, which currently occupy only 20% of the catchment area (A0), flowing to the closing profile, was calculated from the proportion.The A5 scenario is the difference between the nutrient load from the A0 scenario and the sum of the remaining loads from the subsequent variant scenarios (A1–A4). In this way, both the natural background (NBS) and atmospheric deposition (ATS) were taken into account. More

  • in

    Balsam fir (Abies balsamea) needles and their essential oil kill overwintering ticks (Ixodes scapularis) at cold temperatures

    Kilpatrick, A. M. et al. Lyme disease ecology in a changing world: consensus, uncertainty and critical gaps for improving control. Philos. Trans. R. Soc. B-Biol. Sci. 372, 15. https://doi.org/10.1098/rstb.2016.0117 (2017).Article 

    Google Scholar 
    Adenubi, O. T. et al. Pesticidal plants as a possible alternative to synthetic acaricides in tick control: A systematic review and meta-analysis. Ind. Crop. Prod. 123, 779–806. https://doi.org/10.1016/j.indcrop.2018.06.075 (2018).CAS 
    Article 

    Google Scholar 
    Jordan, R. A. & Schulze, T. L. Availability and nature of commercial tick control services in three Lyme disease endemic states. J. Med. Entomol. 57, 807–814. https://doi.org/10.1093/jme/tjz215 (2019).CAS 
    Article 

    Google Scholar 
    Isman, M. B. Botanical insecticides in the twenty-first century – Fulfilling their promise?. Ann. Rev. Entomol. 65, 233–249 (2020).CAS 
    Article 

    Google Scholar 
    Eisen, L. Control of ixodid ticks and prevention of tick-borne diseases in the United States: The prospect of a new Lyme disease vaccine and the continuing problem with tick exposure on residential properties. Ticks Tick-Borne Dis. https://doi.org/10.1016/j.ttbdis.2021.101649 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Santos, A. C. C. et al. Apis mellifera (Insecta: Hymenoptera) in the target of neonicotinoids: A one-way ticket? Bioinsecticides can be an alternative. Ecotoxicol. Environ. Safe. 163, 28–36. https://doi.org/10.1016/j.ecoenv.2018.07.048 (2018).CAS 
    Article 

    Google Scholar 
    Matos, W. B. et al. Potential source of ecofriendly insecticides: Essential oil induces avoidance and cause lower impairment on the activity of a stingless bee than organosynthetic insecticides, in laboratory. Ecotoxicol. Environ. Safe. 209, 111764. https://doi.org/10.1016/j.ecoenv.2020.111764 (2021).CAS 
    Article 

    Google Scholar 
    Gashout, H. A., Guzman-Novoa, E., Goodwin, P. H. & Correa-Benítez, A. Impact of sublethal exposure to synthetic and natural acaricides on honey bee (Apis mellifera) memory and expression of genes related to memory. J. Insect Physiol. 121, 104014. https://doi.org/10.1016/j.jinsphys.2020.104014 (2020).CAS 
    Article 
    PubMed 

    Google Scholar 
    Eisen, L. & Dolan, M. C. Evidence for personal protective measures to reduce human contact with Blacklegged ticks and for environmentally based control methods to suppress host-seeking Blacklegged ticks and reduce infection with Lyme disease spirochetes in tick vectors and rodent reservoirs. J. Med. Entomol. 53, 1063–1092. https://doi.org/10.1093/jme/tjw103 (2016).Article 
    PubMed 

    Google Scholar 
    Dyer, M. C., Requintina, M. D., Berger, K. A., Puggioni, G. & Mather, T. N. Evaluating the effects of minimal risk natural products for control of the tick, Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 58, 390–397. https://doi.org/10.1093/jme/tjaa188 (2021).CAS 
    Article 
    PubMed 

    Google Scholar 
    Schulze, T. L. & Jordan, R. A. Synthetic pyrethroid, natural product, and entomopathogenic fungal acaricide product formulations for sustained early season suppression of host-seeking Ixodes scapularis (Acari: Ixodidae) and Amblyomma americanum nymphs. J. Med. Entomol. 58, 814–820. https://doi.org/10.1093/jme/tjaa248 (2021).CAS 
    Article 
    PubMed 

    Google Scholar 
    Bharadwaj, A., Stafford, K. C. & Behle, R. W. Efficacy and environmental persistence of nootkatone for the control of the Blacklegged tick (Acari: Ixodidae) in residential landscapes. J. Med. Entomol. 49, 1035–1044. https://doi.org/10.1603/me11251 (2012).Article 
    PubMed 

    Google Scholar 
    Pavela, R. & Sedlák, P. Post-application temperature as a factor influencing the insecticidal activity of the essential oil from Thymus vulgaris. Ind. Crop. Prod. 113, 46–49 (2018).CAS 
    Article 

    Google Scholar 
    Brunner, J. L., Killilea, M. & Ostfeld, R. S. Overwintering survival of nymphal Ixodes scapularis (Acari: Ixodidae) under natural conditions. J. Med. Entomol. 49, 981–987. https://doi.org/10.1603/me12060 (2012).Article 
    PubMed 

    Google Scholar 
    Chown, S. L. & Nicolson, S. W. Insect Physiol. Ecol. (Oxford University Press, 2004).Ogden, N. H., Beard, C. B., Ginsberg, H. S. & Tsao, J. I. Possible effects of climate change on Ixodid ticks and the pathogens they transmit: Predictions and observations. J. Med. Entomol. 58, 1536–1545 (2021).Article 

    Google Scholar 
    Ballard, K. & Bone, C. Exploring spatially varying relationships between Lyme disease and land cover with geographically weighted regression. Appl. Geo. 127, 102383 (2021).Article 

    Google Scholar 
    Neelakanta, G., Sultana, H., Fish, D., Anderson, J. F. & Fikrig, E. Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold. J. Clin. Invest. 120, 3179–3190. https://doi.org/10.1172/jci42868 (2010).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Adamo, S. A. Animals have a Plan B: how insects deal with the dual challenge of predators and pathogens. J. Comp. Physiol. B-Biochem. Syst. Environ. Physiol. 190, 381–390. https://doi.org/10.1007/s00360-020-01282-5 (2020).Article 

    Google Scholar 
    Adamo, S. A. How insects protect themselves against combined starvation and pathogen challenges, and the implications for reductionism. Comp. Biochem. Physiol. B-Biochem. Molec. Biol. https://doi.org/10.1016/j.cbpb.2021.110564 (2021).Article 

    Google Scholar 
    Linske, M. A. et al. Impacts of deciduous leaf litter and snow presence on nymphal Ixodes scapularis (Acari: Ixodidae) overwintering survival in coastal New England, USA. Insects https://doi.org/10.3390/insects10080227 (2019).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Burtis, J. C., Fahey, T. J. & Yavitt, J. B. Survival and energy use of Ixodes scapularis nymphs throughout their overwintering period. Parasitol. 146, 781–790. https://doi.org/10.1017/s0031182018002147 (2019).Article 

    Google Scholar 
    Boehnke, D., Gebhardt, R., Petney, T. & Norra, S. On the complexity of measuring forests microclimate and interpreting its relevance in habitat ecology: the example of Ixodes ricinus ticks. Parasit. Vectors 10, 549. https://doi.org/10.1186/s13071-017-2498-5 (2017).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Lindsay, L. R. et al. Survival and development of Ixodes scapularis (Acari, Ixodidae) under various climatic conditions in Ontario, Canada. J. Med. Entomol. 32, 143–152. https://doi.org/10.1093/jmedent/32.2.143 (1995).CAS 
    Article 
    PubMed 

    Google Scholar 
    Lindsay, L. R. et al. Survival and development of the different life stages of Ixodes scapularis (Acari: Ixodidae) held within four habitats on Long Point, Ontario, Canada. J. Med. Entomol. 35, 189–199. https://doi.org/10.1093/jmedent/35.3.189 (1998).CAS 
    Article 
    PubMed 

    Google Scholar 
    Ginsberg, H. S. et al. Woodland type and spatial distribution of nymphal Ixodes scapularis (Acari: Ixodidae). Environ. Entomol. 33, 1266–1273. https://doi.org/10.1603/0046-225x-33.5.1266 (2004).Article 

    Google Scholar 
    Clow, K. M. et al. The influence of abiotic and biotic factors on the invasion of Ixodes scapularis in Ontario, Canada. Ticks Tick-Borne Dis. 8, 554–563. https://doi.org/10.1016/j.ttbdis.2017.03.003 (2017).Article 
    PubMed 

    Google Scholar 
    Natural Resources Canada. Balsam fir, (2015).Khatchikian, C. E. et al. Recent and rapid population growth and range expansion of the Lyme disease tick vector, Ixodes scapularis North America. Evolution 69, 1678–1689. https://doi.org/10.1111/evo.12690 (2015).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Pichette, A., Larouche, P. L., Lebrun, M. & Legault, J. Composition and antibacterial activity of Abies balsamea essential oil. Phytotherapy Res. 20, 371–373 (2006).CAS 
    Article 

    Google Scholar 
    Poaty, B., Lahlah, J., Porqueres, F. & Bouafif, H. Composition, antimicrobial and antioxidant activities of seven essential oils from the North American boreal forest. World J. Microbiol. Biotechnol. 31, 907–919. https://doi.org/10.1007/s11274-015-1845-y (2015).CAS 
    Article 
    PubMed 

    Google Scholar 
    Beasley, T. M. & Schumacker, R. E. Multiple regression approach to analyzing contingency tables: Post hoc and planned comparison procedures. J. Exp. Ed. 64, 79–93. https://doi.org/10.1080/00220973.1995.9943797 (1995).Article 

    Google Scholar 
    Canon, L., Deslauriers, A., Mshvildadze, V. & Pichette, A. Volatile compounds in the foliage of balsam fir analyzed by static headspace gas chromotography (HS-GS): An example of the spruce budworm defoliation effect in the boreal forest of Quebec, Canada. Microchem. J. 110, 587–590 (2013).Article 

    Google Scholar 
    Faraone, N., MacPherson, S. & Hillier, N. K. Behavioral responses of Ixodes scapularis tick to natural products: development of novel repellents. Exp. Appl. Acarol. 79, 195–207. https://doi.org/10.1007/s10493-019-00421-0 (2019).CAS 
    Article 
    PubMed 

    Google Scholar 
    McMillan, L. E., Miller, D. W. & Adamo, S. A. Eating when ill is risky: immune defense impairs food detoxification in the caterpillar Manduca sexta. J. Exp. Biol. 221, 173336 (2018).
    Google Scholar 
    Gazave, E., Chevillon, C., Lenormand, T., Marquine, M. & Raymond, M. Dissecting the cost of insecticide resistance genes during the overwintering period of the mosquito Culex pipiens. Heredity 87, 441–448 (2001).CAS 
    Article 

    Google Scholar 
    Lalouette, L., Williams, C. M., Hervant, F., Sinclair, B. J. & Renault, D. Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations. Comp. Biochem. Physiol. A 158, 229–234 (2011).CAS 
    Article 

    Google Scholar 
    Clark, D. D. Lower temperature limits for activity of several Ixodid ticks: Effects of body size and rate of temperature change. J. Med. Entomol. 32, 449–452 (1995).CAS 
    Article 

    Google Scholar 
    Carroll, J. F. & Kramer, M. Winter activity of Ixodes scapularis (Acari : Ixodidae) and the operation of deer-targeted tick control devices in Maryland. J. Med. Entomol. 40, 238–244. https://doi.org/10.1603/0022-2585-40.2.238 (2003).CAS 
    Article 
    PubMed 

    Google Scholar 
    Ginsberg, H. S. et al. Environmental factors affecting survival of immature Ixodes scapularis and implications for geographical distribution of Lyme disease: the climate/behavior hypothesis. PLoS ONE 12, e0168723. https://doi.org/10.1371/journal.pone.0168723 (2017).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Quadros, D. G., Johnson, T. L., Whitney, T. R., Oliver, J. D. & Chavez, A. S. O. Plant-derived natural compounds for tick pest control in livestock and wildlife: Pragmatism or utopia?. Insects https://doi.org/10.3390/insects11080490 (2020).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Ogendo, J. et al. Biocontrol potential of selected plant essential oil constituents as fumigants of insect pests attacking stored food commodities. Health 10, 287–318 (2011).
    Google Scholar 
    Panella, N. A., Karchesy, J., Maupin, G. O., Malan, J. C. & Piesman, J. Susceptibility of immature Ixodes scapularis (Acari: Ixodidae) to plant-derived acaricides. J. Med. Entomol. 34, 340–345 (1997).CAS 
    Article 

    Google Scholar 
    Rosado-Aguilar, J. A. et al. Plant products and secondary metabolites with acaricide activity against ticks. Vet. Parasitol. 238, 66–76. https://doi.org/10.1016/j.vetpar.2017.03.023 (2017).CAS 
    Article 
    PubMed 

    Google Scholar 
    Jaenson, T. G. T., Carboui, S. & Palsson, K. Repellency of oils of lemon eucalyptus, geranium, and lavender and the mosquito repellent MyggA natural to Ixodes ricinus (Acari : Ixodidae) in the laboratory and field. J. Med. Entomol. 43, 731–736. https://doi.org/10.1603/0022-2585(2006)43[731:Rooole]2.0.Co;2 (2006).CAS 
    Article 
    PubMed 

    Google Scholar 
    Eigbrett, C. Natural Sourcing Organic Essential Oils Oxford, Connecticut, USA, .praannaturals.com/downloads/msds/SDS_Organic_Essential_Oil_Fir_Balsam_Canada.pdf (2016).Schulze, T. L. et al. Efficacy of granular deltamethrin against Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) nymphs. J. Med. Entomol. 38, 344–346. https://doi.org/10.1603/0022-2585-38.2.344 (2001).CAS 
    Article 
    PubMed 

    Google Scholar 
    Elias, S. P. et al. Effect of a botanical acaricide on Ixodes scapularis (Acari: Ixodidae) and nontarget arthropods. J. Med. Entomol. 50, 126–136. https://doi.org/10.1603/me12124 (2013).Article 
    PubMed 

    Google Scholar 
    Burtis, J. C., Yavitt, J. B., Fahey, T. J. & Ostfeld, R. S. Ticks as soil-dwelling arthropods: an intersection between disease and soil ecology. J. Med. Entomol. 56, 1555–1564. https://doi.org/10.1093/jme/tjz116 (2019).Article 
    PubMed 

    Google Scholar 
    Burtis, J. C., Ostfeld, R. S., Yavitt, J. B. & Fahey, T. J. The relationship between soil arthropods and the overwinter survival of Ixodes scapularis (Acari: Ixodidae) under manipulated snow cover. J. Med. Entomol. 53, 225–229. https://doi.org/10.1093/jme/tjv151 (2016).CAS 
    Article 
    PubMed 

    Google Scholar 
    Guerra, M. et al. Predicting the risk of Lyme disease: Habitat suitability for Ixodes scapularis in the north central United States. Emerg. Infect. Dis. 8, 289–297. https://doi.org/10.3201/eid0803.010166 (2002).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Bunnell, J. E., Price, S. D., Das, A., Shields, T. M. & Glass, G. E. Geographic information systems and spatial analysis of adult Ixodes scapularis (Acari: Ixodidae) in the Middle Atlantic region of the USA. J. Med. Entomol. 40, 570–576. https://doi.org/10.1603/0022-2585-40.4.570 (2003).Article 
    PubMed 

    Google Scholar 
    Lubelczyk, C. B. et al. Habitat associations of Ixodes scapularis (Acari: Ixodidae) in Maine. Environ. Entomol. 33, 900–906. https://doi.org/10.1603/0046-225x-33.4.900 (2004).Article 

    Google Scholar 
    Killilea, M. E., Swei, A., Lane, R. S., Briggs, C. J. & Ostfeld, R. S. Spatial dynamics of Lyme disease: A review. EcoHealth 5, 167–195. https://doi.org/10.1007/s10393-008-0171-3 (2008).Article 
    PubMed 

    Google Scholar 
    Stafford, K. C. Survival of immature Ixodes scapularis (Acari: Ixodidae) at different relative humidities. J. Med. Entomol. 31, 310–314 (1994).Article 

    Google Scholar 
    Bertrand, M. R. & Wilson, M. L. Microclimate-dependent survival of unfed adult Ixodes scapularis (Acari: Ixodidae) in Nature: Life cycle and study design implications. J. Med. Entomol. 33, 619–627 (1996).CAS 
    Article 

    Google Scholar 
    Lindsay, L. R. et al. Microclimate and habitat in relation to Ixodes scapularis (Acari: Ixodidae) populations on Long Point, Ontario, Canada. J. Med. Entomol. 36, 255–262. https://doi.org/10.1093/jmedent/36.3.255 (1999).CAS 
    Article 
    PubMed 

    Google Scholar 
    Thompson, C., Spielman, A. & Krause, P. J. Coinfecting deer-associated zoonoses: Lyme disease, babesiosis, and ehrlichiosis. Clin. Infect. Dis. 33, 676–685 (2001).CAS 
    Article 

    Google Scholar 
    Hinckley, A. F. et al. effectiveness of residential acaricides to prevent Lyme and other tick-borne diseases in humans. J. Infect. Dis. 214, 182–188. https://doi.org/10.1093/infdis/jiv775 (2016).Article 
    PubMed 

    Google Scholar 
    Keesing, F. et al. Effects of Ttck-control interventions on tick abundance, human encounters with Ttcks, and incidence of tickborne diseases in residential neighborhoods, New York, USA. Emerg. Infect. Dis. 28, 957–966. https://doi.org/10.3201/eid2805.211146 (2022).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Hayes, L. E., Scott, J. A. & Stafford, K. C. Influences of weather on Ixodes scapularis nymphal densities at long-term study sites in Connecticut. Ticks Tick-Borne Dis. 6, 258–266. https://doi.org/10.1016/j.ttbdiS.2015.01.006 (2015).Article 
    PubMed 

    Google Scholar 
    Rand, P. W. et al. Trial of a minimal-risk botanical compound to control the vector tick of Lyme disease. J. Med. Entomol. 47, 695–698 (2010).CAS 
    Article 

    Google Scholar 
    United Nations. Convention on Biological Diversity. (1992).Convention on International Trade in Endangered Species of Wild Fauna and Flora. (1973).Burtis, J. C. Method for the efficient deployment and recovery of Ixodes scapularis (Acari: Ixodidae) nymphs and engorged larvae from field microcosms. J. Med. Entomol. 54, 1778–1782. https://doi.org/10.1093/jme/tjx157 (2017).Article 
    PubMed 

    Google Scholar 
    Nova Scotia Department of Natural Resources and Renewables Trees of the Acadian Forest (2021). More

  • in

    Combining multi-marker metabarcoding and digital holography to describe eukaryotic plankton across the Newfoundland Shelf

    Lombard, F. et al. Consistent quantitative observations of planktonic ecosystems. Front. Mar. Sci. 6, 196. https://doi.org/10.3389/fmars.2019.00196 (2019).Article 

    Google Scholar 
    Sieracki, M. E., et al. Optical plankton imaging and analysis systems for ocean observation. Proceedings of OceanObs’09: Sustained Ocean Observations and Information for Society, 878–885 (2010). https://doi.org/10.5270/OceanObs09.cwp.81.Irisson, J.-O., Ayata, S.-D., Lindsay, D. J., Karp-Boss, L. & Stemmann, L. Machine learning for the study of plankton and marine snow from images. Ann. Rev. Mar. Sci. 14(1), 277. https://doi.org/10.1146/annurev-marine-041921-013023 (2022).Article 
    PubMed 

    Google Scholar 
    Mars Brisbin, M., Brunner, O. D., Grossmann, M. M. & Mitarai, S. Paired high-throughput, in situ imaging and high-throughput sequencing illuminate acantharian abundance and vertical distribution. Limnol. Oceanogr. 65(12), 2953–2965. https://doi.org/10.1002/lno.11567 (2020).ADS 
    CAS 
    Article 

    Google Scholar 
    Benfield, M. et al. RAPID: Research on automated plankton identification. Oceanography 20(2), 172–187. https://doi.org/10.5670/oceanog.2007.63 (2007).Article 

    Google Scholar 
    Colin, S. et al. Quantitative 3D-imaging for cell biology and ecology of environmental microbial eukaryotes. Elife 6, e26066. https://doi.org/10.7554/eLife.26066 (2017).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Kim, M. K. Principles and techniques of digital holographic microscopy. J. Photonics Energy. 1, 018005. https://doi.org/10.1117/6.0000006 (2010).Article 

    Google Scholar 
    Tahara, T., Quan, X., Otani, R., Takaki, Y. & Matoba, O. Digital holography and its multidimensional imaging applications: A review. Microscopy 67(2), 55–67. https://doi.org/10.1093/jmicro/dfy007 (2018).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Jericho, S. K., Garcia-Sucerquia, J. F. W., Jericho, M. H. & Kreuzer, H. J. Submersible digital in-line holographic microscope. Rev. Sci. Instrum. 77(4), 043706. https://doi.org/10.1063/1.2193827 (2006).ADS 
    CAS 
    Article 

    Google Scholar 
    Bochdansky, A. B., Jericho, M. H. & Herndl, G. J. Development and deployment of a point-source digital inline holographic microscope for the study of plankton and particlesto a depth of 6000 m. Limnol. Oceanogr: Methods 11, 28–40 (2013).Article 

    Google Scholar 
    Yourassowsky, C. & Dubois, F. High throughput holographic imaging-in-flow for the analysis of a wide plankton size range. Opt. Express 22(6), 6661. https://doi.org/10.1364/OE.22.006661 (2014).ADS 
    Article 
    PubMed 

    Google Scholar 
    Jericho, M. H. & Kreuzer, H. J. Point source digital in-line holographic microscopy. In Coherent Light Microscopy (eds Ferraro, P. et al.) 3–30 (Springer, 2011).Chapter 

    Google Scholar 
    Kanka, M., Riesenberg, R. & Kreuzer, H. J. Reconstruction of high-resolution holographic microscopic images. Opt. Lett. 34(8), 1162. https://doi.org/10.1364/OL.34.001162 (2009).ADS 
    CAS 
    Article 
    PubMed 

    Google Scholar 
    Jericho, M. H., Kreuzer, H. J., Kanka, M. & Riesenberg, R. Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy. Appl. Opt. 51(10), 1503. https://doi.org/10.1364/AO.51.001503 (2012).ADS 
    CAS 
    Article 
    PubMed 

    Google Scholar 
    Wu, Y. & Ozcan, A. Lensless digital holographic microscopy and its applications in biomedicine and environmental monitoring. Methods 136, 4–16 (2018).CAS 
    Article 

    Google Scholar 
    Sun, H. et al. digital holography for studies of marine plankton. Philos. Trans. R. Soc. A. 366, 1789–1806 (2008).ADS 
    CAS 
    Article 

    Google Scholar 
    Bianco, V. et al. microplastic identification via holographic imaging and machine learning. Adv. Intell. Syst. 2(2), 1900153. https://doi.org/10.1002/aisy.201900153 (2020).Article 

    Google Scholar 
    Guo, B. et al. Automated plankton classification from holographic imagery with deep convolutional neural networks. Limnol. Oceanogr. 19(1), 21–36. https://doi.org/10.1002/lom3.10402 (2021).Article 

    Google Scholar 
    Nayak, A. R., Malkiel, E., McFarland, M. N., Twardowski, M. S. & Sullivan, J. M. A Review of holography in the aquatic sciences: In situ characterization of particles, plankton, and small scale biophysical interactions. Front. Mar. Sci. 7, 572147. https://doi.org/10.3389/fmars.2020.572147 (2021).Article 

    Google Scholar 
    Di Bella, J. M., Bao, Y., Gloor, G. B., Burton, J. P. & Reid, G. High throughput sequencing methods and analysis for microbiome research. J. Microbiol. Methods 95(3), 401–414. https://doi.org/10.1016/j.mimet.2013.08.011 (2013).CAS 
    Article 
    PubMed 

    Google Scholar 
    Stoeck, T. et al. Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol. Ecol. 19, 21–31. https://doi.org/10.1111/j.1365-294X.2009.04480.x (2010).CAS 
    Article 
    PubMed 

    Google Scholar 
    de Vargas, C. et al. Eukaryotic plankton diversity in the sunlit ocean. Science 348(6237), 1261605–1261605. https://doi.org/10.1126/science.1261605 (2015).CAS 
    Article 
    PubMed 

    Google Scholar 
    Lima-Mendez, G. et al. Determinants of community structure in the global plankton interactome. Science 348(6237), 1262073–1262073. https://doi.org/10.1126/science.1262073 (2015).CAS 
    Article 
    PubMed 

    Google Scholar 
    Santoferrara, L. et al. Perspectives from ten years of protist studies by high-throughput metabarcoding. J. Eukaryot. Microbiol. 67(5), 612–622. https://doi.org/10.1111/jeu.12813 (2020).Article 
    PubMed 

    Google Scholar 
    Eickbush, T. H. & Eickbush, D. G. Finely orchestrated movements: evolution of the ribosomal RNA genes. Genetics 175(2), 477–485. https://doi.org/10.1534/genetics.107.071399 (2007).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Kirkham, A. R. et al. Basin-scale distribution patterns of photosynthetic picoeukaryotes along an Atlantic Meridional Transect: Marine photosynthetic picoeukaryote community structure. Environ. Microbiol. 13(4), 975–990. https://doi.org/10.1111/j.1462-2920.2010.02403.x (2011).CAS 
    Article 
    PubMed 

    Google Scholar 
    Decelle, J. et al. PhytoREF: A reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy. Mol. Ecol. Resour. 15(6), 1435–1445. https://doi.org/10.1111/1755-0998.12401 (2015).CAS 
    Article 
    PubMed 

    Google Scholar 
    Leray, M. & Knowlton, N. Censusing marine eukaryotic diversity in the twenty-first century. Phil. Trans. R. Soc. B. 371(1702), 20150331. https://doi.org/10.1098/rstb.2015.0331 (2016).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Cowart, D. A. et al. Metabarcoding is powerful yet still blind: A comparative analysis of morphological and molecular surveys of seagrass communities. PLoS ONE 10(2), e0117562. https://doi.org/10.1371/journal.pone.0117562 (2015).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Stefanni, S. et al. Multi-marker metabarcoding approach to study mesozooplankton at basin scale. Sci. Rep. 8(1), 12085. https://doi.org/10.1038/s41598-018-30157-7 (2018).ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Pappalardo, P. et al. The role of taxonomic expertise in interpretation of metabarcoding studies. ICES J. Mar. Sci. https://doi.org/10.1093/icesjms/fsab082 (2021).Article 

    Google Scholar 
    Gloor, G. B., Macklaim, J. M., Pawlowsky-Glahn, V. & Egozcue, J. J. Microbiome datasets are compositional: And this is not optional. Front. Microbiol. 8, 2224. https://doi.org/10.3389/fmicb.2017.02224 (2017).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Zhu, F., Massana, R., Not, F., Marie, D. & Vaulot, D. Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene. FEMS Microbiol. Ecol. 52(1), 79–92. https://doi.org/10.1016/j.femsec.2004.10.006 (2005).CAS 
    Article 
    PubMed 

    Google Scholar 
    Sargent, E. C. et al. Evidence for polyploidy in the globally important diazotroph Trichodesmium. FEMS Microbiol. Lett. 363(21), 244. https://doi.org/10.1093/femsle/fnw244 (2016).CAS 
    Article 

    Google Scholar 
    Gong, W. & Marchetti, A. Estimation of 18S gene copy number in marine eukaryotic plankton using a next-generation sequencing approach. Front. Mar. Sci. 6, 219. https://doi.org/10.3389/fmars.2019.00219 (2019).Article 

    Google Scholar 
    Biard, T. et al. Biogeography and diversity of collodaria (radiolaria) in the global ocean. ISME J. 11, 1331–1344 (2017).Article 

    Google Scholar 
    Callahan, B. J., McMurdie, P. J. & Holmes, S. P. Exact sequence variants should replace operational taxonomic units in marker-gene data analysis. ISME J. 11(12), 2639–2643. https://doi.org/10.1038/ismej.2017.119 (2017).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Behrenfeld, M. J. et al. The North Atlantic aerosol and marine ecosystem study (NAAMES): Science motive and mission overview. Front. Mar. Sci. 6, 122. https://doi.org/10.3389/fmars.2019.00122 (2019).Article 

    Google Scholar 
    Bolaños, L. M. et al. Seasonality of the microbial community composition in the North Atlantic. Front. Mar. Sci. 8, 624164. https://doi.org/10.3389/fmars.2021.624164 (2021).Article 

    Google Scholar 
    Aitchison, J. The statistical analysis of compositional data. J. R. Stat. Soc. B 44(2), 139–160. https://doi.org/10.1111/j.2517-6161.1982.tb01195.x (1982).MathSciNet 
    Article 
    MATH 

    Google Scholar 
    Decelle, J. & Not, F. Acantharia. ELS, 1–10 (2015). https://doi.org/10.1002/9780470015902.a0002102.pub2.Yu, L., An, Y. & Cai, L. Numerical reconstruction of digital holograms with variable viewing angles. Opt. Express 10(22), 1250. https://doi.org/10.1364/OE.10.001250 (2002).ADS 
    Article 
    PubMed 

    Google Scholar 
    Della Penna, A. & Gaube, P. Overview of (sub)mesoscale Ocean dynamics for the NAAMES field program. Front. Mar. Sci. 6, 384. https://doi.org/10.3389/fmars.2019.00384 (2019).Article 

    Google Scholar 
    Sverdrup, H. U. Oceanography for Meteorologists (Prentice Hall, 1942).Book 

    Google Scholar 
    Mahadevan, A. The impact of submesoscale physics on primary productivity of plankton. Annu. Rev. Mar. Sci. 8(1), 161–184. https://doi.org/10.1146/annurev-marine-010814-015912 (2016).ADS 
    Article 

    Google Scholar 
    Fratantoni, P. S. & Pickart, R. S. The Western North Atlantic shelfbreak current system in summer. J. Phys. Oceanogr. 37(10), 2509–2533. https://doi.org/10.1175/JPO3123.1 (2007).ADS 
    Article 

    Google Scholar 
    Bolaños, L. M. et al. Small phytoplankton dominate western North Atlantic biomass. ISME J. 14(7), 1663–1674. https://doi.org/10.1038/s41396-020-0636-0 (2020).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Kramer, S. J., Siegel, D. A. & Graff, J. R. Phytoplankton community composition determined from co-variability among phytoplankton pigments from the NAAMES field campaign. Front. Mar. Sci. 7, 215. https://doi.org/10.3389/fmars.2020.00215 (2020).Article 

    Google Scholar 
    Faure, E. et al. Mixotrophic protists display contrasted biogeographies in the global ocean. ISME J. 13(4), 1072–1083. https://doi.org/10.1038/s41396-018-0340-5 (2019).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Fratantoni, P. S. & McCartney, M. S. Freshwater export from the labrador current to the North Atlantic Current at the tail of the grand banks of Newfoundland. Deep Sea Res. I. 57(2), 258–283. https://doi.org/10.1016/j.dsr.2009.11.006 (2010).Article 

    Google Scholar 
    Torti, A., Lever, M. A. & Jørgensen, B. B. Origin, dynamics, and implications of extracellular DNA pools in marine sediments. Mar. Genom. 24, 185–196. https://doi.org/10.1016/j.margen.2015.08.007 (2015).Article 

    Google Scholar 
    Jian, C., Salonen, A. & Korpela, K. Commentary: How to count our microbes? The effect of different quantitative microbiome profiling approaches. Front. Cell. Infect. Microbiol. 11, 627910. https://doi.org/10.3389/fcimb.2021.627910 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Djurhuus, A. et al. Evaluation of marine zooplankton community structure through environmental DNA metabarcoding: Metabarcoding zooplankton from eDNA. Limnol. Oceanogr. Methods 16(4), 209–221. https://doi.org/10.1002/lom3.10237 (2018).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    del Campo, J. et al. The others: Our biased perspective of eukaryotic genomes. Trends Ecol. Evol. 29(5), 252–259. https://doi.org/10.1016/j.tree.2014.03.006 (2014).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Karst, S. M. et al. Retrieval of a million high-quality, full-length microbial 16S and 18S rRNA gene sequences without primer bias. Nat. Biotech. 36(2), 190–195. https://doi.org/10.1038/nbt.4045 (2018).CAS 
    Article 

    Google Scholar 
    Johnson, J. S. et al. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nat. Commun. 10(1), 5029. https://doi.org/10.1038/s41467-019-13036-1 (2019).ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Callahan, B. J. et al. High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution. Nucleic Acids Res. 47(18), e103–e103. https://doi.org/10.1093/nar/gkz569 (2019).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Lin, Y., Gifford, S., Ducklow, H., Schofield, O. & Cassar, N. Towards quantitative microbiome community profiling using internal standards. Appl. Environ. Microbiol. 85(5), 18. https://doi.org/10.1128/AEM.02634-18 (2019).Article 

    Google Scholar 
    Vogt, M. et al. Global marine plankton functional type biomass distributions: Phaeocystis spp. Earth Syst. Sci. Data 5, 405–443. https://doi.org/10.5194/essdd-5-405-2012 (2012).ADS 
    Article 

    Google Scholar 
    MacNeil, L., Missan, S., Luo, J., Trappenberg, T. & LaRoche, J. Plankton classification with high-throughput submersible holographic microscopy and transfer learning. BMC Ecol. Evol. 21(1), 123. https://doi.org/10.1186/s12862-021-01839-0 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Pan, J., del Campo, J. & Keeling, P. J. Reference tree and environmental sequence diversity of labyrinthulomycetes. J. Eukary. Microbiol. 64(1), 88–96. https://doi.org/10.1111/jeu.12342 (2017).Article 

    Google Scholar 
    Bochdansky, A. B., Clouse, M. A. & Herndl, G. J. Eukaryotic microbes, principally fungi and labyrinthulomycetes, dominate biomass on bathypelagic marine snow. ISME J. 11(2), 362–373. https://doi.org/10.1038/ismej.2016.113 (2017).Article 
    PubMed 

    Google Scholar 
    Xie, N., Hunt, D. E., Johnson, Z. I., He, Y. & Wang, G. Annual partitioning patterns of Labyrinthulomycetes protists reveal their multifaceted role in marine microbial food webs. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.01652-20 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Walcutt, N. L. et al. Assessment of holographic microscopy for quantifying marine particle size and concentration. Limnol. Oceanogr. Methods 3, 10379. https://doi.org/10.1002/lom3.10379 (2020).Article 

    Google Scholar 
    Axler, K. et al. Fine-scale larval fish distributions and predator-prey dynamics in a coastal river-dominated ecosystem. Mar. Ecol. Prog. Ser. 650, 37–61. https://doi.org/10.3354/meps13397 (2020).ADS 
    Article 

    Google Scholar 
    Trudnowska, E. et al. Marine snow morphology illuminates the evolution of phytoplankton blooms and determines their subsequent vertical export. Nat. Commun. 12(1), 2816. https://doi.org/10.1038/s41467-021-22994-4 (2021).ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    González, P. et al. Automatic plankton quantification using deep features. J. Plankton Res. 41(4), 449–463. https://doi.org/10.1093/plankt/fbz023 (2019).Article 

    Google Scholar 
    Briseño-Avena, C. et al. Three-dimensional cross-shelf zooplankton distributions off the Central Oregon Coast during anomalous oceanographic conditions. Prog. Oceanogr. 188, 102436. https://doi.org/10.1016/j.pocean.2020.102436 (2020).Article 

    Google Scholar 
    Biard, T. et al. In situ imaging reveals the biomass of giant protists in the global ocean. Nature 532, 504–507 (2016).ADS 
    CAS 
    Article 

    Google Scholar 
    Orenstein, E. C. et al. The scripps plankton camera system: A framework and platform for in situ microscopy. Limnol. Oceanogr. Methods 18(11), 681–695. https://doi.org/10.1002/lom3.10394 (2020).Article 

    Google Scholar 
    Fowler, B. L. et al. Dynamics and functional diversity of the smallest phytoplankton on the Northeast US Shelf. PNAS 117(22), 12215–12221. https://doi.org/10.1073/pnas.1918439117 (2020).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Tréguer, P. et al. Influence of diatom diversity on the ocean biological carbon pump. Nat. Geosci. 11(1), 27–37. https://doi.org/10.1038/s41561-017-0028-x (2018).ADS 
    CAS 
    Article 

    Google Scholar 
    Ryabov, A. et al. Shape matters: The relationship between cell geometry and diversity in phytoplankton. Ecol. Lett. 24(4), 847–861. https://doi.org/10.1111/ele.13680 (2021).MathSciNet 
    Article 
    PubMed 

    Google Scholar 
    Keeling, P. J. & del Campo, J. marine protists are not just big bacteria. Curr. Biol. 27(11), R541–R549. https://doi.org/10.1016/j.cub.2017.03.075 (2017).CAS 
    Article 
    PubMed 

    Google Scholar 
    Sgubin, G., Swingedouw, D., Drijfhout, S., Mary, Y. & Bennabi, A. Abrupt cooling over the North Atlantic in modern climate models. Nat. Commun. 8(1), 14375. https://doi.org/10.1038/ncomms14375 (2017).CAS 
    Article 
    PubMed Central 

    Google Scholar 
    Desbruyères, D., Chafik, L. & Maze, G. A shift in the ocean circulation has warmed the subpolar North Atlantic Ocean since 2016. Commun. Earth Environ. 2(1), 48. https://doi.org/10.1038/s43247-021-00120-y (2021).ADS 
    Article 

    Google Scholar 
    Mitchell, M. R. et al. Atlantic zone monitoring program protocol. Can. Tech. Rep. Hydrogr. Ocean Sci. 223, 1–23 (2002).
    Google Scholar 
    Li, W. K. W., Glen Harrison, W. & Head, E. J. H. Coherent assembly of phytoplankton communities in diverse temperate ocean ecosystems. Proc. R. Soc. B. 273(1596), 1953–1960. https://doi.org/10.1098/rspb.2006.3529 (2006).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Richardson, P. L. Florida current, gulf stream, and labrador current. In Encyclopedia of Ocean Sciences (ed. Steele, J. H.) 1054–1064 (Academic Press, 2001). https://doi.org/10.1006/rwos.2001.0357.Chapter 

    Google Scholar 
    Henson, S. A., Dunne, J. P. & Sarmiento, J. L. Decadal variability in North Atlantic phytoplankton blooms. J. Geophys. Res. 114(C4), C04013. https://doi.org/10.1029/2008JC005139 (2009).ADS 
    CAS 
    Article 

    Google Scholar 
    Han, G., Lu, Z., Wang, Z., Helbig, J. & Chen, N. Seasonal variability of the labrador current and shelf circulation off Newfoundland. J. Geophys. Res. 113, 10. https://doi.org/10.1029/2007JC004376 (2008).Article 

    Google Scholar 
    Pante, E. & Simon-Bouhet, B. marmap: A package for importing, plotting and analyzing bathymetric and topographic data in R. PLoS ONE 8(9), e73051. https://doi.org/10.1371/journal.pone.0073051 (2013).ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Kelley, D. “The Oce Package” In Oceanographic Analysis with R 91–101 (Springer, 2018).Book 

    Google Scholar 
    Oksanen, J., et al. vegan: Community Ecology Package. R package version 2.5-7 (2020). https://CRAN.R-project.org/package=vegan.Tomas, C. R. Identifying Marine Phytoplankton (Academic Press Inc, 1997).
    Google Scholar 
    Comeau, A. M., Li, W. K. W., Tremblay, J. -É., Carmack, E. C. & Lovejoy, C. Arctic ocean microbial community structure before and after the 2007 record sea ice minimum. PLoS ONE 6(11), e27492. https://doi.org/10.1371/journal.pone.0027492 (2011).ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Parada, A. E., Needham, D. M. & Fuhrman, J. A. Every base matters: Assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples: Primers for marine microbiome studies. Environ. Microbiol. 18(5), 1403–1414. https://doi.org/10.1111/1462-2920.13023 (2016).CAS 
    Article 
    PubMed 

    Google Scholar 
    Walters, W. et al. Improved bacterial 16S rRNA gene (V4 and V4–5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. MSystems https://doi.org/10.1128/mSystems.00009-15 (2016).Article 
    PubMed 

    Google Scholar 
    Comeau, A. M., Douglas, G. M. & Langille, M. G. I. Microbiome helper: A custom and streamlined workflow for microbiome research. MSystems 2(1), e00127-e216. https://doi.org/10.1128/mSystems.00127-16 (2017).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotech. 37(8), 852–857. https://doi.org/10.1038/s41587-019-0209-9 (2019).CAS 
    Article 

    Google Scholar 
    Amir, A. et al. Deblur rapidly resolves single-nucleotide community sequence patterns. MSystems 2(2), e00191-e216. https://doi.org/10.1128/mSystems.00191-16 (2017).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Guillou, L. et al. The protist ribosomal reference database (PR2): A catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucleic Acids Res. 41(D1), D597–D604. https://doi.org/10.1093/nar/gks1160 (2013).CAS 
    Article 
    PubMed 

    Google Scholar 
    Mohsen, A., Park, J., Chen, Y.-A., Kawashima, H. & Mizuguchi, K. Impact of quality trimming on the efficiency of reads joining and diversity analysis of Illumina paired-end reads in the context of QIIME1 and QIIME2 microbiome analysis frameworks. BMC Bioinform. 20(1), 581. https://doi.org/10.1186/s12859-019-3187-5 (2019).Article 

    Google Scholar 
    Bokulich, N. A. et al. Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome 6(1), 90. https://doi.org/10.1186/s40168-018-0470-z (2018).MathSciNet 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Quast, C. et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 41(D1), D590–D596. https://doi.org/10.1093/nar/gks1219 (2012).CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    R Core Team. R: A Language and Environment for Statistical Computing. (R Foundation for Statistical Computing, 2021). https://www.R-project.org/.McMurdie, P. J. & Holmes, S. phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8(4), e61217. https://doi.org/10.1371/journal.pone.0061217 (2013).ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Willis, A. & Bunge, J. Estimating diversity via frequency ratios: estimating diversity via ratios. Biometrics 71(4), 1042–1049. https://doi.org/10.1111/biom.12332 (2015).MathSciNet 
    Article 
    PubMed 
    MATH 

    Google Scholar 
    Willis, A. D. Rarefaction, alpha diversity, and statistics. Front. Microbiol. 10, 2407. https://doi.org/10.3389/fmicb.2019.02407 (2019).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Quinn, T. P. et al. A field guide for the compositional analysis of any-omics data. GigaScience 8(9), 107. https://doi.org/10.1093/gigascience/giz107 (2019).CAS 
    Article 

    Google Scholar 
    Silverman, J. D., Roche, K., Mukherjee, S. & David, L. A. Naught all zeros in sequence count data are the same. Comput. Struct. Biotech. J. 18, 2789–2798. https://doi.org/10.1016/j.csbj.2020.09.014 (2020).CAS 
    Article 

    Google Scholar 
    Anderson, M. J. A new method for non-parametric multivariate analysis of variance. Austral. Ecol. 26, 32–46 (2001).
    Google Scholar  More

  • in

    Why the ocean virome matters

    Kyoto University microbiome researcher Hiroyuki Ogata says that the recent work2,3 further connects RNA viruses and the carbon pump, which affects the Earth’s biogeochemical cycles and thus its climate. And it sheds light on the diversity, evolution and ecology of RNA viruses, which has not previously been possible through applying the techniques of traditional DNA-based metagenomics. The team found many new lineages at the phylum-level by using “highly sensitive” computational approaches.It’s possible to assess the ecosystem impact of viruses by inferring auxiliary metabolic genes (AMGs). AMGs hint at the ways RNA viruses manipulate the physiology of their hosts as they seek to maximize production of more virus through the host. As Jian explains, labs have identified a variety of AMGs that are encoded by DNA viruses and, he says, it’s “well-recognized” that AMGs probably play a role in marine ecosystems. It was unknown if AMGs could be found in RNA viruses, which the recent Science paper2 has now established, he says. Jian sees this work as providing “a very important foundational dataset” for exploring questions connected to AMGs. “In my opinion, if more long-sequence or complete marine RNA virus genomes can be obtained in the future, and they can be further connected with specific hosts, it will greatly promote the understanding of the ecological impact of RNA viruses in the oceans.”To tease out AMGs, the scientists used a variety of tools, such as viral identification software for both DNA and RNA viruses, says Wainaina. The ones for DNA viruses are available on Cyverse, and the protocols for the tools from the Sullivan lab are on protocols.io. One method for RNA viruses is in progress and will be soon available on Cyverse, he says. DNA viral identification tools include VirSorter2, a pipeline for identifying viral sequence from metagenomics data, and the protocol for using this and other tools are also on protocols.io. To identify AMGs from viral sequence that had been identified through VirSorter, the team used use DRAM-v, a software tool from the lab of microbiome researcher Kelly Wrighton at Colorado State University. Her group had created Distilled and Refined Annotation of Metabolism (DRAM), a framework to resolve metabolic information from microbial data. The companion tool DRAM-v is for viruses and can be applied to metagenomic data sets for annotating metagenomics-based assembled genomes, for example through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, and to contiguous viral sequences identified by VirSorter.The hunt for AMGs is one instance in which the team needed to determine in each case whether a sequence was likely ‘stolen’ from host cells, says Dominguez-Huerta. RNA viral genomes are less than 40 kilobases long and usually have complicated genomic organization, both in a structural genomics sense related to the physical arrangement of genes along the viral genome and in a functional sense in terms of transcription and translation: there are overlapping genes, frameshifts and more, all of which makes this kind of annotation difficult. And sometimes information in the annotation databases is wrong and indicates that a match is cellular when it is in fact viral. Thus, to find AMGs, “we don’t have a defined clean methodology automated in a pipeline yet,” he says. It remains a time-consuming task. Assigning putative function to the protein sequences encoded by AMGs also involves checking the literature and comparing different annotation sources.Dominguez-Huerta says he and the team were glad they could assemble AMG functionalities to suggest the range of ways in which RNA viruses manipulate the metabolisms of their hosts—from photosynthesis to central carbon metabolism to vacuolar digestion and RNA repair. This overview let them see how some AMGs are repeated across different viruses across the oceans. Finding AMGs in long-read sequence is what he calls a “fire test” for the lab. To avoid ‘false AMGs’ from unreliable matches, they use BLASTP, the Basic Alignment Search Tool that compares a protein query sequence to a protein database.“I am fascinated by the ability of viruses to metabolic reprogram not only their hosts but more importantly at the ecosystem level,” says Wainaina. It is probable that the AMGs the team identified “are a central cog in microbial metabolism networks.” Current and future modeling efforts will hopefully provide insights into the ecosystem roles of viruses—both DNA viruses and RNA viruses—and on a global scale both within the ocean ecosystem and beyond.Host inference is challenging, says Dominguez-Huerta, because, for example, viruses with RNA genomes do not share genetic information with their host genomic DNA the way dsDNA viruses do when they infect bacteria. That means there is no clear signal to be derived from the host genome to help one guess the possible host. But sometimes RNA viruses do integrate into host genomes, and those, likely more accidental, events were sufficient for the scientists to capture some signal to infer hosts. “We also performed statistical co-occurrence analytics using abundances to infer the hosts with certain success,” he says.Unlike dsDNA viruses, RNA viruses infect mostly eukaryotes, from protists and fungi to invertebrates and fish larvae; only a minority infect bacteria. Overall, the team has been able to capture “a picture of dsDNA viruses infecting prokaryotes and RNA viruses infecting eukaryotes in the oceans, complementing each other in their marine hosts,” says Dominguez-Huerta. The fact that the scientists can infer “that RNA viruses can steal genes from the host,” in the form of AMGs, to then reprogram host metabolism matters not only as scientists complete the picture of how viruses directly tune the activity of hosts during infection, but also in regard to how this influences biogeochemical cycles, he says. “We think that these AMGs are incorporated into the RNA virus genomes from cellular mRNA transcripts by non-homologous recombination,” he says. This gives, in his view, a new picture of RNA viruses, which, despite their small genome sizes, can squeeze in protein-coding genes. Such proteins could be sufficient to boost the production of virus particles per infected cell, perhaps increasing viral fitness in the difficult conditions of the oligotrophic open ocean and letting the viruses better propagate in the environment.More generally, says Dominguez-Huerta, capturing RNA from ocean samples is difficult, because RNA is physically fragile and degrades rapidly. When digging into metatranscriptomic data, which include the RNA from plankton and RNA from other organisms, less than 1% of this RNA is likely to be viral RNA, he says. Previously, some labs have first purified RNA from samples, enriched it for replicating RNA viruses and then applied a method called dsRNA-seq to recover dsRNA virus sequence and replicate sequences from single-stranded RNA viruses. For future ocean RNA virus projects, he says that the lab is currently working on a wet-lab method to purify RNA virus particles from seawater to solve the challenges of obtaining viral RNA for analysis. More

  • in

    Stochastic models of Mendelian and reverse transcriptional inheritance in state-structured cancer populations

    Pienta, K. J., Hammarlund, E. U., Austin, R. H., Axelrod, R., Brown, J. S. & Amend, S. R. Cancer cells employ an evolutionarily conserved polyploidization program to resist therapy. In Seminars in Cancer Biology, 1–15 (2020).Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2020. CA A Cancer J. Clin. 70(1), 7–30 (2020).Article 

    Google Scholar 
    Duesberg, P. & Rasnick, D. Aneuploidy, the somatic mutation that makes cancer a species of its own. Cell Motil. Cytoskelet. 47(2), 81–107 (2000).CAS 
    Article 

    Google Scholar 
    Hanahan, D. & Weinberg, R. A. Leading edge review hallmarks of cancer: The next generation. Cell 144, 646–674 (2011).CAS 
    PubMed 
    Article 

    Google Scholar 
    Amend, S. R. et al. Polyploid giant cancer cells: Unrecognized actuators of tumorigenesis, metastasis, and resistance. Prostate 79(13), 1489–1497 (2019).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Pienta, K. J. et al. Convergent evolution, evolving evolvability, and the origins of lethal cancer. Mol. Cancer Res. 18(6), 801–810 (2020).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Pienta, K. J., Hammarlund, E. U., Axelrod, R., Brown, J. S. & Amend, S. R. Poly-aneuploid cancer cells promote evolvability, generating lethal cancer. Evol. Appl. 13(7), 1626–1634 (2020).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Roychowdhury, S. et al. Personalized oncology through integrative high-throughput sequencing: A pilot study. Sci. Transl. Med. 3(111), 1–12 (2011).Article 
    CAS 

    Google Scholar 
    Kuczler, M. D., Olseen, A. M., Pienta, K. J. & Amend, S. R. ROS-induced cell cycle arrest as a mechanism of resistance in polyaneuploid cancer cells (PACCs). Prog. Biophys. Mol. Biol. 165, 3–7 (2021).CAS 
    PubMed 
    Article 

    Google Scholar 

    Brown, R. L. What evolvability really is. Br. J. Philos. Sci.65(3), 549–572 (2014).MathSciNet 
    Article 

    Google Scholar 
    Crother, B. I. & Murray, C. M. Early usage and meaning of evolvability. Ecol. Evol. 9(7), 3784–3793 (2019).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Payne, J. L. & Wagner, A. The causes of evolvability and their evolution. Nat. Rev. Genet. 20, 24–38 (2019).CAS 
    PubMed 
    Article 

    Google Scholar 
    Pigliucci, M. Is evolvability evolvable?. Genetics 9, 75–82 (2008).CAS 
    PubMed 

    Google Scholar 
    Sniegowski, P. D. & Murphy, H. A. Evolvability. Curr. Biol. 16, R831–R834 (2006).CAS 
    PubMed 
    Article 

    Google Scholar 
    Kostecka, L. G., Pienta, K. J. & Amend, S. R. Polyaneuploid cancer cell dormancy: Lessons from evolutionary phyla. Front. Ecol. Evol. 9, 439 (2021).Article 

    Google Scholar 
    Rajaraman, R., Rajaraman, M. M., Rajaraman, S. R. & Guernsey, D. L. Neosis–a paradigm of self-renewal in cancer. Cell Biol. Int. 29(12), 1084–1097 (2005).CAS 
    PubMed 
    Article 

    Google Scholar 
    Rajaraman, R., Guernsey, D. L., Rajaraman, M. M. & Rajaraman, S. R. Neosis–A parasexual somatic reduction division in cancer. Int. J. Hum. Genet. 7(1), 29–48 (2007).CAS 
    Article 

    Google Scholar 
    Sundaram, M., Guernsey, D. L., Rajaraman, M. M. & Rajaraman, R. Neosis: A novel type of cell division in cancer. Cancer Biol. Ther. 3(2), 207–218 (2004).CAS 
    PubMed 
    Article 

    Google Scholar 
    Gatenby, R. A., Cunningham, J. J. & Brown, J. S. Evolutionary triage governs fitness in driver and passenger mutations and suggests targeting never mutations. Nat. Commun. 5(1), 1–9 (2014).Article 

    Google Scholar 
    Bukkuri, A. & Brown, J. S. Evolutionary game theory: Darwinian dynamics and the G function approach. MDPI Games 12(4), 1–19 (2021).MathSciNet 
    MATH 

    Google Scholar 
    Lopez-Sánchez, L. M. et al. CoCl2, a mimic of hypoxia, induces formation of polyploid giant cells with stem characteristics in colon cancer. PLoS ONE 9(6), e99143 (2014).ADS 
    PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 
    Mittal, K. et al. Multinucleated polyploidy drives resistance to Docetaxel chemotherapy in prostate cancer. Br. J. Cancer 116(9), 1186–1194 (2017).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Niu, N., Mercado-Uribe, I. & Liu, J. Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells. Oncogene 36(34), 4887–4900 (2017).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ogden, A., Rida, P. C. G., Knudsen, B. S., Kucuk, O. & Aneja, R. Docetaxel-induced polyploidization may underlie chemoresistance and disease relapse. Cancer Lett. 367, 89–92 (2015).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Puig, P. E. et al. Tumor cells can escape DNA-damaging cisplatin through DNA endoreduplication and reversible polyploidy. Cell Biol. Int. 32(9), 1031–1043 (2008).CAS 
    PubMed 
    Article 

    Google Scholar 
    Zhang, S. et al. Generation of cancer stem-like cells through the formation of polyploid giant cancer cells. Oncogene 33(1), 116–128 (2014).CAS 
    PubMed 
    Article 

    Google Scholar 
    Lin, K. C. et al. The role of heterogeneous environment and docetaxel gradient in the emergence of polyploid, mesenchymal and resistant prostate cancer cells. Clin. Exp. Metastasis 36(2), 97–108 (2019).PubMed 
    Article 

    Google Scholar 
    Lin, K.-C. et al. Epithelial and mesenchymal prostate cancer cell population dynamics on a complex drug landscape. Converg. Sci. Phys. Oncol. 3(4), 045001 (2017).PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 
    Boe, L. Mechanism for induction of adaptive mutations in Escherichia coli. Mol. Microbiol. 4(4), 597–601 (1990).CAS 
    PubMed 
    Article 

    Google Scholar 
    Cairns, J. Mutation and cancer: The antecedents to our studies of adaptive mutation. Genetics 148(4), 1433–1440 (1998).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Hall, B. G. Adaptive mutagenesis: A process that generates almost exclusively beneficial mutations. Genetica 102, 109 (1998).PubMed 
    Article 

    Google Scholar 
    Waddington, C. H. Genetic assimilation of an acquired character. Evolution 7(2), 118–126 (1953).Article 

    Google Scholar 
    Waddington, C. H. Genetic assimilation. Adv. Genet. 10, 257–293 (1961).CAS 
    PubMed 
    Article 

    Google Scholar 
    Jablonka, E. V. A. & Raz, G. A. L. Transgenerational epigenetic inheritance: Prevalence, mechanisms, and implications for the study of heredity and evolution. Q. Rev. Biol. 84(2), 131–176 (2009).PubMed 
    Article 

    Google Scholar 
    Steele, E. J. & Pollard, J. W. Hypothesis: Somatic hypermutation by gene conversion via the error prone DNA(longrightarrow )RNA(longrightarrow )DNA information loop. Mol. Immunol. 24(6), 667–673 (1987).CAS 
    PubMed 
    Article 

    Google Scholar 
    Steele, E. J. Somatic hypermutation in immunity and cancer: Critical analysis of strand-biased and codon-context mutation signatures. DNA Repair 45, 1–24 (2016).CAS 
    PubMed 
    Article 

    Google Scholar 
    Steele, E. J. Somatic Selection and Adaptive Evolution (Springer, US, 1979).
    Google Scholar 
    Steele, E. J., Lindley, R. A. & Blanden, R. V. Lamarck’s Signature (Perseus Books, 1998).
    Google Scholar 
    Foster, P. L. Adaptive mutation: Implications for evolution. Bioessays 22, 1067–1074 (2000).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    McCutcheon, J. P. & Moran, N. A. Extreme genome reduction in symbiotic bacteria. Nat. Rev. Microbiol. 10(1), 13–26 (2012).CAS 
    Article 

    Google Scholar 
    Badyaev, A. V. Stress-induced variation in evolution: From behavioural plasticity to genetic assimilation. Proc. R. Soc. B Biol. Sci. 272, 877–886 (2005).Article 

    Google Scholar 
    Bateman, K. G. The genetic assimilation of four venation phenocopies. J. Genet. 56(3), 443–474 (1959).Article 

    Google Scholar 
    Milkman, R. D. The genetic basis of natural variation. VI. Selection of a crossveinless strain of Drosophila by phenocopying at high temperature. Genetics 51(1), 87 (1965).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Waddington, C. H. Genetic assimilation of the bithorax phenotype. Evolution 10(1), 1–13 (1956).Article 

    Google Scholar 
    Godoy, O., Saldaña, A., Fuentes, N., Valladares, F. & Gianoli, E. Forests are not immune to plant invasions: Phenotypic plasticity and local adaptation allow Prunella vulgaris to colonize a temperate evergreen rainforest. Biol. Invasions 13(7), 1615–1625 (2011).Article 

    Google Scholar 
    Schlichting, C. D. & Wund, M. A. Phenotypic plasticity and epigenetic marking: An assessment of evidence for genetic accommodation. Evolution 68(3), 656–672 (2014).PubMed 
    Article 

    Google Scholar 
    Otaki, J. M., Hiyama, A., Iwata, M. & Kudo, T. Phenotypic plasticity in the range-margin population of the lycaenid butterfly Zizeeria maha. BMC Evol. Biol. 10(1), 1–13 (2010).Article 

    Google Scholar 
    Aubret, F. & Shine, R. Genetic assimilation and the postcolonization erosion of phenotypic plasticity in island tiger snakes. Curr. Biol. 19(22), 1932–1936 (2009).CAS 
    PubMed 
    Article 

    Google Scholar 
    Losos, J. B., Irschick, D. J. & Schoener, T. W. Adaptation and constraint in the evolution of specialization of Bahamian Anolis lizards. Evolution 48(6), 1786–1798 (1994).PubMed 
    Article 

    Google Scholar 
    Losos, J. B. et al. Evolutionary implications of phenotypic plasticity in the hindlimb of the lizard Anolis sagrei. Evolution 54(1), 301–305 (2000).CAS 
    PubMed 

    Google Scholar 
    Sword, G. A. Density-dependent warning coloration. Nature 397(6716), 217 (1999).ADS 
    CAS 
    Article 

    Google Scholar 
    Sword, G. A. A role for phenotypic plasticity in the evolution of aposematism. Proc. R. Soc. B Biol. Sci. 269(1501), 1639–1644 (2002).Article 

    Google Scholar 
    Clausen, J. & Hiesey, W. M. The balance between coherence and variation in evolution. Proc. Natl. Acad. Sci. 46(4), 494–506 (1960).ADS 
    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Gurevitch, J. Variation in leaf dissection and leaf energy budgets among populations of Achillea from an altitudinal gradient. Am. J. Bot. 75(9), 1298–1306 (1988).Article 

    Google Scholar 
    Gurevitch, J. & Schuepp, P. H. Boundary layer properties of highly dissected leaves: An investigation using an electrochemical fluid tunnel. Plant Cell Environ. 13(8), 783–792 (1990).Article 

    Google Scholar 
    Gurevitch, J. Sources of variation in leaf shape among two populations of Achillea lanulosa. Genetics 130(2), 385–394 (1992).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Foster, P. L. Stress-induced mutagenesis in bacteria. Crit. Rev. Biochem. Mol. Biol. 42(5), 373–397 (2007).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Soppa, J. Polyploidy in archaea and bacteria: About desiccation resistance, giant cell size, long-term survival, enforcement by a eukaryotic host and additional aspects. Microb. Physiol. 24, 409–419 (2014).ADS 
    CAS 
    Article 

    Google Scholar 
    Bastide, A. & David, A. The ribosome, (slow) beating heart of cancer (stem) cell. Oncogenesis 7(4), 1–13 (2018).CAS 
    Article 

    Google Scholar 
    Cairns, J., Overbaugh, J. & Miller, S. The origin of mutants. Nature 335, 142–145 (1988).ADS 
    CAS 
    PubMed 
    Article 

    Google Scholar 
    Foster, P. L. Adaptive mutation: The uses of adversity. Annu. Rev. Microbiol. 47, 467–504. https://doi.org/10.1146/annurev.mi.47.100193.002343 (2003).Article 

    Google Scholar 
    Lenski, R. E. & Mittler, J. E. The directed mutation controversy and neo-Darwinism. Science 259(5092), 188–194 (1993).ADS 
    CAS 
    PubMed 
    Article 

    Google Scholar 
    Lenski, R. E. & Sniegowski, P. D. “Adaptive mutation’’: The debate goes on. Science 269, 285–288 (1995).ADS 
    CAS 
    PubMed 
    Article 

    Google Scholar 
    Noller, H. F., Hoffarth, V. & Zimniak, L. Unusual resistance of peptidyl transferase to protein extraction procedures. Science 256(5062), 1416–1419 (1992).ADS 
    CAS 
    PubMed 
    Article 

    Google Scholar 
    Pribis, J. P. et al. Gamblers: An antibiotic-induced evolvable cell subpopulation differentiated by reactive-oxygen-induced general stress response. Mol. Cell 74(4), 785–800 (2019).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Silvera, D., Formenti, S. C. & Schneider, R. J. Translational control in cancer. Nat. Rev. Cancer 10(4), 254–266 (2010).CAS 
    PubMed 
    Article 

    Google Scholar 
    Shcherbakov, D. et al. Ribosomal mistranslation leads to silencing of the unfolded protein response and increased mitochondrial biogenesis. Commun. Biol. 2(1), 1–16 (2019).CAS 
    Article 

    Google Scholar 
    Truitt, M. L. & Ruggero, D. New frontiers in translational control of the cancer genome. Nat. Rev. Cancer 16(5), 288–304 (2016).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Alphey, L. S., Crisanti, A., Randazzo, F. & Akbari, O. S. Opinion: Standardizing the definition of gene drive. Proc. Natl. Acad. Sci. USA 117(49), 30864 (2020).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Champer, J., Buchman, A. & Akbari, O. S. Cheating evolution: Engineering gene drives to manipulate the fate of wild populations. Nat. Rev. Genet. 17, 146–159 (2016).CAS 
    PubMed 
    Article 

    Google Scholar 
    Champer, S. E. et al. Modeling CRISPR gene drives for suppression of invasive rodents using a supervised machine learning framework. PLOS Comput. Biol. 17(12), e1009660 (2021).ADS 
    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Deredec, A., Burt, A. & Godfray, H. C. J. The population genetics of using homing endonuclease genes in vector and pest management. Genetics 179(4), 2013–2026 (2008).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Heffel, M. G. & Finnigan, G. C. Mathematical modeling of self-contained CRISPR gene drive reversal systems. Sci. Rep. 9(1), 1–10 (2019).Article 
    CAS 

    Google Scholar 
    Leftwich, P. T. et al. Recent advances in threshold-dependent gene drives for mosquitoes. Biochem. Soc. Trans. 46, 1203–1212 (2018).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Nijhout, H. F., Kudla, A. M. & Hazelwood, C. C. Genetic assimilation and accommodation: Models and mechanisms. Curr. Top. Dev. Biol. 141, 337–369 (2021).PubMed 
    Article 

    Google Scholar 
    Noble, C., Adlam, B., Church, G. M., Esvelt, K. M. & Nowak, M. A. Current CRISPR gene drive systems are likely to be highly invasive in wild populations. eLife 7, e33423 (2018).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Novozhilov, A. S., Karev, G. P. & Koonin, E. V. Mathematical modeling of evolution of horizontally transferred genes. Mol. Biol. Evol. 22(8), 1721–1732 (2005).CAS 
    PubMed 
    Article 

    Google Scholar 
    Pigliucci, M. & Murren, C. J. Perspective: Genetic assimilation and a possible evolutionary paradox: Can macroevolution sometimes be so fast as to pass us by?. Evolution 57, 1455–1464 (2003).PubMed 
    Article 

    Google Scholar 
    Hammerstein, P. Darwinian adaptation, population genetics and the streetcar theory of evolution. J. Math. Biol. 34(5–6), 511–532 (1996).CAS 
    PubMed 
    MATH 
    Article 

    Google Scholar 
    Dieckmann, U. Coevolutionary Dynamics of Stochastic Replicator Systems (Central Library of the Research Center Jülich, 1994).
    Google Scholar 
    Dieckmann, U., Marrow, P. & Law, R. Evolutionary cycling in predator-prey interactions: population dynamics and the red queen. J. Theor. Biol. 176(1), 91–102 (1995).ADS 
    CAS 
    PubMed 
    Article 

    Google Scholar 
    Dieckmann, U. & Law, R. The dynamical theory of coevolution: a derivation from stochastic ecological processes. J. Math. Biol. 34, 579–612 (1996).MathSciNet 
    CAS 
    PubMed 
    MATH 
    Article 

    Google Scholar 
    Metz, J. A. J., Nisbet, R. M. & Geritz, S. A. H. How should we define ‘fitness’ for general ecological scenarios?. Trends Ecol. Evol. 7(6), 198–202 (1992).CAS 
    PubMed 
    Article 

    Google Scholar 
    Goldschmidt, R. Some aspects of evolution. Science 78(2033), 539–547 (1933).ADS 
    CAS 
    PubMed 
    Article 

    Google Scholar 
    Vincent, T. L., Cohen, Y. & Brown, J. S. Evolution via strategy dynamics. Theor. Popul. Biol. 44(2), 149–176 (1993).MATH 
    Article 

    Google Scholar 
    Bell, G. Evolutionary rescue. Annu. Rev. Ecol. Evol. Syst. 48, 605–627 (2017).Article 

    Google Scholar  More

  • in

    Biogeographic implication of temperature-induced plant cell wall lignification

    Körner, C. The cold range limit of trees. Trends Ecol. Evo. 36, 979–989 (2021).Article 

    Google Scholar 
    Körner, C. Alpine Treelines (Springer, 2012).Miehe, G., Miehe, S., Vogel, J., Co, S. & Duo, L. Highest treeline in the northern hemisphere found in southern Tibet. Mt. Res. Dev. 27, 169–173 (2007).Article 

    Google Scholar 
    Hoch, G. & Körner, C. Growth, demography and carbon relations of Polylepis trees at the world’s highest treeline. Funct. Ecol. 19, 941–951 (2005).Article 

    Google Scholar 
    von Humboldt, A. & Bonpland, A. Ideen zu einer Geographie der Pflanzen nebst einem Naturgemälde der Tropenländer: auf Beobachtungen und Messungen gegründet, welche vom 10ten Grade nördlicher bis zum 10ten Grade südlicher Breite, in den Jahren 1799, 1800, 1801, 1802 und 1803 angestellt worden sind. Tübingen, Bey F.G. Cotta (1807).Körner, C. Climatic treelines: conventions, global patterns, causes. Erdkunde 61, 315–324 (2007).Article 

    Google Scholar 
    Piermattei, A., Crivellaro, A., Carrer, M. & Urbinati, C. The “blue ring”: anatomy and formation hypothesis of a new tree-ring anomaly in conifers. Trees Struct. Funct. 29, 613–620 (2015).CAS 
    Article 

    Google Scholar 
    Körner, C. et al. Life at 0 °C: the biology of the alpine snowbed plant Soldanella pulsatilla. Alp. Bot. 129, 63–80 (2019).Article 

    Google Scholar 
    Crivellaro, A. & Büntgen, U. New evidence of thermally-constraint plant cell wall lignification. Trends Plant Sci. 24, 322–324 (2020).Article 
    CAS 

    Google Scholar 
    Büntgen, U. et al. Temperature-induced recruitment pulses of Arctic dwarf shrub communities. J. Ecol. 103, 489–501 (2015).Article 

    Google Scholar 
    Dolezal, J. et al. Vegetation dynamics at the upper elevational limit of vascular plants in Himalaya. Sci. Rep. 6, 24881 (2016).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ryan, M. G. & Yoder, B. J. Hydraulic limits to tree height and tree growth. Biosci 47, 235–242 (1997).Article 

    Google Scholar 
    Koch, G. W., Sillett, S. C., Jennings, G. M. & Davis, S. D. The limits to tree height. Nature 428, 851–854 (2004).CAS 
    PubMed 
    Article 

    Google Scholar 
    Körner, C. Alpine Plant Life: Functional Plant Ecology of High Mountain Ecosystems (Springer, 2003).Scherrer, D. & Körner, C. Infra-red thermometry of alpine landscapes challenges climatic warming projections. Glob. Change Biol. 16, 2602–2613 (2010).
    Google Scholar 
    Begum, S., Nakaba, S., Yamagishi, Y., Oribe, Y. & Funada, R. Regulation of cambial activity in relation to environmental conditions: understanding the role of temperature in wood formation of trees. Physiol. Planta 147, 46–54 (2013).CAS 
    Article 

    Google Scholar 
    Plomion, C., Leprovost, G. & Stokes, A. Wood formation in trees. Plant Physiol. 127, 1513–1523 (2001).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Rossi, S., Deslauriers, A., Anfodillo, T. & Carraro, V. Evidence of threshold temperatures for xylogenesis in conifers at high altitudes. Oecologia 152, 1–12 (2007).PubMed 
    Article 

    Google Scholar 
    Moura, J. C. M. S., Bonine, C. A. V., Viana, J. O. F., Dornelas, M. C. & Mazzafera, P. Abiotic and biotic stresses and changes in the lignin content and composition in plants. J. Integr. Plant Biol. 52, 360–376 (2010).CAS 
    PubMed 
    Article 

    Google Scholar 
    Weng, J. K. & Chapple, C. The origin and evolution of lignin biosynthesis. N. Phytol. 187, 273–285 (2010).CAS 
    Article 

    Google Scholar 
    Niklas, K. J., Cobb, E. D. & Matas, A. J. The evolution of hydrophobic cell wall biopolymers: from algae to angiosperms. J. Exp. 68, 5261–5269 (2017).CAS 

    Google Scholar 
    Popper, Z. A. et al. Evolution and diversity of plant cell walls: from algae to flowering plants. Annu. Rev. Plant Biol. 62, 567–590 (2011).CAS 
    PubMed 
    Article 

    Google Scholar 
    Piquemal, J. et al. Down regulation of cinnamoyl CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant J. 13, 71–83 (1998).CAS 
    Article 

    Google Scholar 
    Renault, H., Werck-Reichhart, D. & Weng, J.-K. Harnessing lignin evolution for biotechnological applications. Curr. Opin. Biotechnol. 56, 105–111 (2019).CAS 
    PubMed 
    Article 

    Google Scholar 
    Schenk, H. J., Espino, S., Rich-Cavazos, S. M. & Jansen, S. From the sap’s perspective: The nature of vessel surfaces in angiosperm xylem. Am. J. Bot. 105, 172–185 (2018).CAS 
    PubMed 
    Article 

    Google Scholar 
    Polo, C. C. et al. Correlations between lignin content and structural robustness in plants revealed by X-ray ptychography. Sci. Rep. 10, 6023 (2020).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Meents, M. J., Watanabe, Y. & Samuels, A. L. The cell biology of secondary cell wall biosynthesis. Ann. Bot. 121, 1107–1125 (2018).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Campbell, M. M. & Sederoff, R. R. Variation in lignin content and composition (mechanisms of control and implications for the genetic improvement of plants). Plant Physiol. 110, 3–13 (1996).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Schweingruber, F. H. & Büntgen, U. What is ‘wood’ – An anatomical re-definition. Dendrochronologia 31, 187–191 (2013).Article 

    Google Scholar 
    Ellenberg, H. & Mueller-Dombois, D. A key to Raunkiaer plant life forms with revised subdivisions. Ber. Geobot. Inst. ETH Z.ürich. 37, 56–73 (1967).
    Google Scholar 
    Kim, W. J., Campbell, A. G. & Koch, P. Chemical variation in Lodgepole pine with latitude, elevation, and diameter class. Prod. J. 39, 7–12 (1989).CAS 

    Google Scholar 
    Gindl, W., Grabner, M. & Wimmer, R. The influence of temperature on latewood lignin content in treeline Norway spruce compared with maximum density and ring width. Trees, Struct. Funct. 14, 409–414 (2000).Article 

    Google Scholar 
    Schenker, G., Lens, A., Körner, C. & Hoch, G. Physiological minimum temperatures for root growth in seven common European broad-leaved tree species. Tree Physiol. 34, 302–313 (2014).PubMed 
    Article 

    Google Scholar 
    Nagelmüller, S., Hiltbrunner, E. & Körner, C. Low temperature limits for root growth in alpine species are set by cell differentiation. AoB Plants 9, plx054 (2017).PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ji, H. et al. The Arabidopsis RCC1 family protein TCF1 regulates freezing tolerance and cold acclimation through modulating lignin biosynthesis. PLoS Gen. 11, e1005471 (2015).Article 
    CAS 

    Google Scholar 
    Büntgen, U. Re-thinking the boundaries of dendrochronology. Dendrochronologia 53, 1–4 (2019).Article 

    Google Scholar 
    Piermattei, A. et al. A millennium-long ‘Blue-Ring’ chronology from the Spanish Pyrenees reveals sever ephemeral summer cooling after volcanic eruptions. Environ. Res. Lett. 15, 124016 (2020).Article 

    Google Scholar 
    Montwé, D., Isaac-Rentin, M., Hamman, A. & Spiecker, H. Cold adaptation recorded in tree rings highlights risks associated with climate change and assisted migration. Nat. Comm. 9, 1574 (2018).Article 
    CAS 

    Google Scholar 
    Barros, J., Serk, H., Granlund, I. & Pesquet, E. The cell biology of lignification in higher plants. Ann. Bot. 115, 1053–1074 (2015).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Hao, Z. & Mohnen, D. A review of xylan and lignin biosynthesis: Foundation for studying Arabidopsis irregular xylem mutants with pleiotropic phenotypes. Cri. Rev. Biochem. Mol. Biol. 49, 212–241 (2014).CAS 
    Article 

    Google Scholar 
    Liu, Q., Luo, L. & Zheng, L. Lignins: biosynthesis and biological functions in plants. Int. J. Mol. Sci. 19, 335 (2018).PubMed Central 
    Article 
    CAS 

    Google Scholar 
    Kumar, M., Campbell, L. & Turner, S. Secondary cell walls: biosynthesis and manipulation. J. Exp. Bot. 67, 515–531 (2016).CAS 
    PubMed 
    Article 

    Google Scholar 
    Mellerowicz, E. J., Baucher, M., Sundberg, B. & Boerjan, W. Unravelling cell wall formation in the woody dicot stem. Plant Mol. Biol. 47, 239–274 (2001).CAS 
    PubMed 
    Article 

    Google Scholar 
    Petit, G., Anfodillo, T., Carraro, V., Grani, F. & Carrer, M. Hydraulic constraints limit height growth in trees at high altitude. N. Phytol. 189, 241–252 (2010).Article 

    Google Scholar 
    Li, L. et al. Combinatorial modification of multiple lignin traits in trees through multigene co-transformation. Proc. Natl Acad. Sci. USA 100, 4939–4944 (2003).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Baldacci-Cresp, F. et al. A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification. Plant J. 102, 1074–1089 (2020).CAS 
    PubMed 
    Article 

    Google Scholar 
    Körner, C. A re-assessment of high elevation treeline positions and their explanation. Oecologia 115, 445–459 (1998).PubMed 
    Article 

    Google Scholar 
    Landolt, E. et al. Flora indicativa: Okologische Zeigerwerte und biologische Kennzeichen zur Flora der Schweiz und der Alpen (Haupt, 2010).Büntgen, U., Psomas, A. & Schweingruber, F. H. Introducing wood anatomical and dendrochronological aspects of herbaceous plants: applications of the Xylem Database to vegetation science. J. Veg. Sci. 25, 967–977 (2014).Article 

    Google Scholar 
    Körner, C. Coldest places on earth with angiosperm plant life. Alp. Bot. 121, 11–22 (2011).Article 

    Google Scholar 
    GBIF.org. GBIF Occurrence Download. https://doi.org/10.15468/dl.ms4hjt (2018).Chamberlain, S., Ram, K. & Hart, T. Spocc: Interface to Specie Occurrence Data Sources, R package v.0.9.0. http://CRAN.R-project.org/package=spocc (2018).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 
    Hijmans, R. J. Raster: geographic data analysis and modelling, R package v.2.2-12. http://CRAN.R-project.org/package=raster (2014).Gärtner, H. et al. A technical perspective in modern tree-ring research – How to overcome dendroecological and wood anatomical challenges. J. Vis. Exp. 97, e52337 (2015).
    Google Scholar 
    Gärtner, H. & Schweingruber, F. H. Microscopic Preparation Techniques for Plant Stem Analysis (Verlag Kessel, 2013).Ghislan, B., Engel, J. & Clair, B. Diversity of anatomical structure of tension wood among 242 tropical tree species. IAWA J. 40, 1–20 (2019).Article 

    Google Scholar 
    Schweingruber, F. H., Börner, A. & Schulze, E. D. Atlas of Stem Anatomy in Herbs, Shrubs and Trees Vol. 1 (Springer, 2011).Schweingruber, F. H., Börner, A. & Schulze, E. D. Atlas of Stem Anatomy in Herbs, Shrubs and Trees Vol. 2 (Springer, 2013).Dolezal, J., Dvorsky, M., Börner, A., Wild, J. & Schweingruber, F. H. Anatomy, Age and Ecology of High Mountain Plants in Ladakh, the Western Himalaya (Springer International Publishing, 2018).Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH image to imageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 
    Ter Braak, C. J. F. & Šmilauer, P. Canoco Reference Manual and User’s Guide: Software 559 for Ordination, Version 5.0 (Cambridge Univ. Press, 2012).Šmilauer, P. & Lepš, J. Multivariate Analysis of Ecological Data Using Canoco 5 (Cambridge Univ. Press, 2014). More