Philippa Kaur

Aagaard-Tillery KM, Grove K, Bishop J, Ke X, Fu Q, McKnight R et al. (2008) Developmental origins of disease and determinants of chromatin structure: maternal diet modifies the primate fetal epigenome. J Mol Endocrinol 41:91–102Article 
CAS 

Google Scholar 
Alekseev V, Lampert W (2001) Maternal control of resting – egg production in Daphnia. Nature 414:899–901Article 
CAS 

Google Scholar 
Anderson OS, Sant KE, Dolinoy DC (2012) Nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and DNA methylation. J Nutritional Biochem 23:853–859Article 
CAS 

Google Scholar 
Agrawal AA, Laforsch C, Tollrian R (1999) Transgenerational induction of defenses in animals and plants. Nature 401:60–63Article 
CAS 

Google Scholar 
Asselman J, De Coninck DIM, Vandegehuchte MB, Jansen M, Decaestecker E, De Meester L, Busshe JV, Vanhaecke L, Janssen CR, De Schamphelaere KAC (2015) Global cytosine methylation in Daphnia magna depends on genotype, environment, and their interaction. Environ Toxicol 34:5
Google Scholar 
Bernardo J (1996) Maternal effects in animal ecology. Am Zool 36:83–105Article 

Google Scholar 
Berger SL (2007) The complex language of chromatin regulation during transcription. Nature 447:407–412Article 
CAS 

Google Scholar 
Bewick AJ, Vogel KJ, Moore AJ, Schmitz RJ (2017) Evolution of DNA methylation across insects. Mol Biol Evol 34:654–665CAS 

Google Scholar 
Bird A (2007) Perceptions of epigenetics. Nature 447:396–398Article 
CAS 

Google Scholar 
Boersma M (1995) The allocation of resources to reproduction in Daphnia galeata: against the odds? Ecology 76(4):121–1261Article 

Google Scholar 
Boersma M (1997) Offspring size in Daphnia: does it pay to be overweight? Hydrobiologia 360:79–88Article 

Google Scholar 
Boycott AE, Diver C (1923) On the inheritance of the sinistrality in Limnea peregra. Proc R Soc Lond B 95:207–213Article 

Google Scholar 
Brett MT (1993) Resource quality effects on Daphnia longispina offspring fitness. J Plankton Res 15(4):403–412Article 

Google Scholar 
Burns CW (1995) Effects of crowding and different food levels on growth and reproductive investment of Daphnia. Oecologia 101:234–244Article 

Google Scholar 
Cameron NM, Shahrokh D, Del Corpo A, Dhir SK, Szyf M, Champagne FA, Meaney MJ (2008) Epigenetic programming of phenotypic variations in reproductive strategies in the rat through maternal care. J Neuroendocrinol 20:795–801Article 
CAS 

Google Scholar 
Champagne FA (2012) Epigenetics and developmental plasticity across species. Dev Psychobiol 55:33–41Article 

Google Scholar 
Chan SY, Vasilopoulou E, Kilby MD (2009) The role of the placenta in thyroid hormone delivery to the fetus. Nat Clin Pract Endocrinol Metab 5:45–54Article 
CAS 

Google Scholar 
Chong S, Whitelaw E (2004) Epigenetic germline inheritance. Curr Opin Genet Dev 14(6):692–696Article 
CAS 

Google Scholar 
Clark J, Garbutt JS, McNally L, Little TJ (2017) Disease spread in age structured populations with maternal age effects. Ecol Lett 20:445–451Article 

Google Scholar 
Colbourne JK, Herbert PDN, Taylor DJ (1997) Evolutionary origins of phenotypic diversity. In: Givnish TJ, Systma KJ (eds) Daphnia in molecular evolution and adaptive radiation. Cambridge University Press. p 163–188Colbourne JK, Pfrender ME, Gilbert D, Thomas WK, Tucker A, Oakley TH et al. (2011) The ecoresponsive genome of Daphnia pulex. Science 331(6017):555–561Article 
CAS 

Google Scholar 
Desmarais KH (1997) Keeping Daphnia out of the surface film with cetyl alcohol. J Plankton Res 19(1):149–154Article 

Google Scholar 
Dorts J, Falisse E, Schoofs E, Flamion E, Kestermont P, Silvestre F (2016) DNA methyltransferases and stress-related genes expression in zebrafish larvae after exposure to heat and copper during reprogramming of DNA methylation. Sci Rep 6:34254Article 
CAS 

Google Scholar 
Ducker GS, Rabinowitz JD (2016) One-carbon metabolism in health and disease. Cell Metab 25:27–42. https://doi.org/10.1016/j.cmet.2016.08Dudycha JL, Brandon CS, Deitz KC (2012) Population genomics of resource exploitation: insights from gene expression profiles of two Daphnia ecotypes fed alternate resources. Ecol Evol 2:329–340Dzialowski EM, Reed WL, Sotherland PR (2009) Effects of egg size on double-crested cormorant (Phalacrocorax auritus) egg composition and hatchling phenotype. Comp Biochem Physiol A Mol Integr Physiol 152:262–267Article 

Google Scholar 
Frost PC, Ebert D, Larson JH, Marcus MA, Wagner ND, Zalewski A (2010) Transgenerational effects of poor elemental food quality on Daphnia magna. Oecologia 162(4):865–872Article 

Google Scholar 
Gabsi F, Glazier DS, Hammers-Wirtz M, Ratte HT, Preuss TG (2014) How to interactive maternal traits and environmental factors determine offspring size in Daphnia magna?. Ann Limnol 50:9–18Article 

Google Scholar 
Garbutt JS, Little TJ (2016) Bigger is better: changes in body size explain a maternal effect of food on offspring disease resistance. Ecol Evolution 7:1403–1409Article 

Google Scholar 
Gibney ER, Nolan CM (2010) Epigenetics and gene expression. Heredity 105:4–13Article 
CAS 

Google Scholar 
Gillis MK, Walsh MR (2019) Individual variation in plasticity dulls transgenerational responses to stress. Funct Ecol 33:1993–2002Glazier DS (1992) Effects of food, genotype, and maternal size and age on offspring investment in Daphnia magna. Ecology 73(3):910–926Article 

Google Scholar 
Gliwicz ZM, Guisande C (1992) Family planning in Daphnia: resistance to starvation in offspring born to mothers grown at different food levels. Oceologia 91:463–467Article 

Google Scholar 
Goos JM, Swain CJ, Munch SB, Walsh MR (2018) Maternal diet and age alter direct and indirect relationships between lifer-history traits across multiple generations. Funct Ecol 33:491–502Article 

Google Scholar 
Goulden CE, Horning LL (1980) Population oscillations and energy reserves in planktonic cladocera and their consequences to competition. Proc Natl Acad Sci USA 77:1716–1720Article 
CAS 

Google Scholar 
Groothuis TG, Schwabl H (2008) Hormone-mediated maternal effects in birds: mechanisms matter but what do we know of them? Philos Trans R Soc Lond B Biol Sci 363:1647–1661Article 
CAS 

Google Scholar 
Guisande C, Gliwicz ZM (1992) Egg size and clutch size in two Daphnia species at different food levels. J Plankton Res 14(7):997–1007Article 

Google Scholar 
Hearn J, Chow FW-N, Barton H, Tung M, Wilson P, Blaxter M et al. (2018) Daphnia magna microRNAs respond to nutritional stress and ageing but are not transgenerational. Mol Ecol 27:1402–1412Article 
CAS 

Google Scholar 
Hearn J, Pearson M, Blaxter M, Wilson PJ, Little TJ (2019) Genome-wide methylation is modified by caloric restriction in Daphnia magna. BCM Genetics 20:197Hearn J, Plenderleith F, Little TJ (2021) DNA methylation differs extensively between strains of the same geographical origin and changes with age in Daphnia magna. Epigenetics Chromatin 14:4. https://doi.org/10.1186/s13072-020-00379-zHead JA (2014) Patterns of DNA methylation in animals: an ecotoxicological perspective. Integr Comp Biol 54:77–86Article 
CAS 

Google Scholar 
Hebert PDN (1981) Obligate asexuality in Daphnia. Am Nate 117:784–789Article 

Google Scholar 
Herman JJ, Sultan SE (2016) DNA methylation mediates genetic variation for adaptive transgenerational plasticity. Proc Biol Sci 283(1838):20160988. https://doi.org/10.1098/rspb.2016.0988Article 
CAS 

Google Scholar 
Hiruta C, Nishida C, Tochinai S (2010) Abortive meiosis in the oogenesis of parthenogenetic Daphnia pulex. Chromosome Res 18:833–840Article 
CAS 

Google Scholar 
Ho DH, Burggren WW (2010) Epigenetics and transgenerational transfer: a physiological perspective. J Exp Biol 213:3–16Article 
CAS 

Google Scholar 
Ho DH (2008) Morphological and physiological developmental consequences of parental effects in the chicken embryo (Gallus gallus domesticus) and the zebrafish larva (Danio rerio). Diss: University of North TexasInnes DJ, Fox CJ, Winsor GL (2000) Avoiding the cost of males in obligately asexual Daphnia pulex (Leydig). Proc: Biol Sci 267(1447):991–997CAS 

Google Scholar 
Jeremias G, Barbosa J, Marques SM, De Schamphelaere KAC, Van Nieuwerburgh F, Deforce D, Gonçalves FJM, Pereira JL, Asselman J (2018) Transgenerational inheritance of dna hypomethylation in Daphnia magna in response to salinity stress. Environ Sci Technol 52(17):10114–10123Article 
CAS 

Google Scholar 
Jian X, Yang W, Zhao S, Liang H, Zhao Y, Chen L et al. (2013) Maternal effects of inducible tolerance against the toxic cyanobacterium Microcystis aeruginosa in the grazer Daphnia carinata. Environ Pollut 178:142–146Article 

Google Scholar 
Keating KI (1985) The influence of vitamin-B12 deficiency on the reproduction of Daphnia-Pulex Leydig (Cladocera). J Crustacean Biol 5:30–136Article 

Google Scholar 
Kleiven OT, Larsson P, Hobaek A (1992) Sexual reproduction in Daphnia magna requires three stimulie. Oikos 65:197–206Article 

Google Scholar 
Kusari F, O’Doherty AM, Hodges NJ, Wojewodzic MW (2017) Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation. Sci Rep 7:11872Article 

Google Scholar 
Kvist J, Athanasio CG, Solari OS, Brown JB, Colbourne JK, Pfrender ME, Mirbahai L (2018) Pattern of DNA methylation in Daphnia: evolutionary perspective. Genome Biol Evolution 10(8):1988–2007Article 
CAS 

Google Scholar 
Lamka GF, Harder AM, Sundaram M, Schwartz TS, Christie MR, DeWoody JA, Willoughby JR (2022) Epigenetics in ecology, evolution, and conservation. Front Ecol Evol 10. https://doi.org/10.3389/fevo.2022.871791LaMontagne JM, McCauley E (2001) Maternal effects in Daphnia: what mothers are telling their offspring and do they listen. Ecol Lett 4:64–71Article 

Google Scholar 
Li Q, Jiang X (2014) Offspring tolerance to toxic Microcystis aeruginosa in Daphnia pulex shaped by maternal food availability and age. Fundam Appl Limnol 185:315–319Article 

Google Scholar 
Mastorci F, Vicentini M, Viltart O, Manghi M, Graiani G, Quaini Fet al. (2009) Long-term effects of prenatal stress: changes in adult cardiovascular regulation and sensitivity to stress. Neurosci Biobehav Rev 33:191–203Article 

Google Scholar 
Mkee D, Ebert D (1996) The interactive effects of temperature, food level and maternal phenotype on offspring size in Daphnia magna. Oecologia 107(2):189–196Article 

Google Scholar 
Mousseau TA, Fox CW (1998) The adaptive significance of maternal effects. Trends Ecol Evol 13:403–407Article 
CAS 

Google Scholar 
Nguyen ND, Matsuura T, Kato Y, Watanabe H (2020) Caloric restriction upregulates the expression of DNMT3.1, lacking the conserved catalytic domain, in Daphnia magna. Genesis 58:12Article 

Google Scholar 
Nguyen ND, Matsuura T, Kato Y, Watanabe H (2021) DNMT3.1 controls trade-offs between growth, reproduction, and life span under starved conditions in Daphnia magna. Sci Rep 11:7326Article 
CAS 

Google Scholar 
Nusslein-Volhard C, Frohnhofer HG, Lehmann R (1987) Determination of anteroposterior polarity in Drosophila. Science 238:1675–1681Article 
CAS 

Google Scholar 
Pieters BJ, Liess M (2006) Maternal nutritional state determines the sensitivity of Daphnia magna offspring to short-term fenvalerate exposure. Aquat Toxicol 76:286–277Article 

Google Scholar 
R Core Team (2021) R: a language and environmental for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/Richards EJ (2006) Inherited epigenetic variation – revisiting soft inheritance. Nat Rev Genet 7:395–401Article 
CAS 

Google Scholar 
Stollewerk A (2010) The water flea Daphnia – a new model system for ecology and evolution? J Biol 9(2):21Article 

Google Scholar 
Sturtevant AH (1923) Inheritance of direction of coiling in Limnea. Science 58:269Article 
CAS 

Google Scholar 
Suter MA, Chen A, Burdine MS, Choudhury M, Harris RA, Lane RH et al. (2012) A maternal high-fat diet modulates fetal SIRT1 histone and protein deacetylase activity in nonhuman primates. FASEB J 26:5106–5114Article 
CAS 

Google Scholar 
Tessier AJ, Consolatti NL (1989) Variation in offspring size in Daphnia and consequences for individual fitness. Oikos 56:269–276Article 

Google Scholar 
Tessier AJ, Consolatti NL (1991) Resource quantity and offspring quality in Daphnia. Ecology 72(2):468–478Article 

Google Scholar 
Trerotola M, Relli V, Simeone P, Alberti S (2015) Epigenetic inheritance and the missing heritability. Hum Genomics 9(1):17. https://doi.org/10.1186/s40246-015-0041-3Article 
CAS 

Google Scholar 
Trijau M, Asselman J, Armant O, Adam-Guillermin C, De Schamphelaere KAC, Alonzo F (2018) Transgenerational DNA methylation changes in Daphnia magna exposed to chronic γ irradiation. Environ Sci Technol 52(7):4331–4339Article 
CAS 

Google Scholar 
Urabe J, Sterner RW (2001) Contrasting effects of different types of resource depletion on life-history traits in Daphnia. Funct Ecol 15:165–174Article 

Google Scholar 
Vandegehuchte MB, Kyndt T, Vanholme B, Haegeman A, Gheysen G, Janssen CR (2009a) Occurrence of DNA methylation in Daphnia magna and influence of multigeneration Cd exposure. Environ Int 35(4):700–706Article 
CAS 

Google Scholar 
Vandegehuchte MB, Lemiere F, Janssen CR (2009b) Quantitative DNA-methylation in Daphnia magna and effects of multigeneration Zn exposure. Comp Biochem Physiol C Toxicol Pharmacol 150:343–348Article 
CAS 

Google Scholar 
Vandegehuchte MB, Janssen CR (2011) Epigenetics and its implications for ecotoxicology. Ecotoxicology 20:607–624Article 
CAS 

Google Scholar 
Vandegehuchte MB, Janssen CR (2014) Epigenetics in an ecotoxicological context. Mutat Res Genet Toxicol Environ Mutagen 764–765:36–45Article 

Google Scholar 
Walsh MR, La Pierre KJ, Post DM (2014) Phytoplankton composition modifies predator-driven life history evolution in Daphnia. Evol Ecol 28:397–411Article 

Google Scholar 
Walsh MR, Cooley F, Biles K, Munch SB (2015) Predator-induced phenotypic plasticity within- and across generations: a challenge for theory? Proc R Soc B Biol Sci 282:20142205Article 

Google Scholar 
Wickham H (2016) ggplot2: elegant graphics for data analysis. Springer-Verlag New York. ISBN 978-3-319-24277-4. https://ggplot2.tidyverse.orgWolf JB, Wade MJ (2009) What are maternal effects (and what are they not)? Philos Trans R Soc Lond B Biol Sci 364(1520):1107–1115Article 

Google Scholar 
Zaffagnini F (1987) Reproduction in Daphnia. Mem Ist Ital Idrobiol 45:245–284
Google Scholar 
Zhao S, Fernald RD (2005) Comprehensive algorithm for quantitative real-time polymerase chain reaction. J Comput Biol 12(8):1045–1062Article 

Google Scholar  More

Listen to the latest from the world of science, with Benjamin Thompson.
Your browser does not support the audio element.

Download MP3

In this episode:00:45 World’s oldest DNA shows that mastodons roamed ancient GreenlandDNA recovered from ancient permafrost has been used to reconstruct what an ecosystem might have looked like two million years ago. Their work suggests that Northern Greenland was much warmer than the frozen desert it is today, with a rich ecosystem of plants and animals.Research Article: Kjær et al.Nature Video: The world’s oldest DNA: Extinct beasts of ancient Greenland08:21 Research HighlightsWhy low levels of ‘good’ cholesterol don’t predict heart disease risk in Black people, and how firework displays affect the flights of geese.Research Highlight: ‘Good’ cholesterol readings can lead to bad results for Black peopleResearch Highlight: New Year’s fireworks chase wild geese high into the sky10:31 Modelling the potential emissions of plasticsWhile the global demand for plastics is growing, the manufacturing and disposal of these ubiquitous materials is responsible for significant CO2 emissions each year. This week, a team have modelled how CO2 emissions could vary in the context of different strategies for mitigating climate change. They reveal how under specific conditions the industry could potentially become a carbon sink.Research Article: Stegmann et al.News and Views: Plastics can be a carbon sink but only under stringent conditionsSubscribe to Nature Briefing, an unmissable daily round-up of science news, opinion and analysis free in your inbox every weekday.Never miss an episode. Subscribe to the Nature Podcast on Apple Podcasts, Google Podcasts, Spotify or your favourite podcast app. An RSS feed for Nature Podcast is available too. More

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

Fernández-Martínez, M. et al. Nutrient availability as the key regulator of global forest carbon balance. Nat. Clim. Chang. 4, 471–476 (2014).Article 
ADS 

Google Scholar 
Wright, S. J. Plant responses to nutrient addition experiments conducted in tropical forests. Ecol. Monogr. 89, e01382 (2019).Article 

Google Scholar 
Levy-Varon, J. H. et al. Tropical carbon sink accelerated by symbiotic dinitrogen fixation. Nat. Commun. 10, 5637 (2019).Article 
ADS 
CAS 

Google Scholar 
Batterman, S. A. et al. Key role of symbiotic dinitrogen fixation in tropical forest secondary succession. Nature 502, 224–227 (2013).Article 
ADS 
CAS 

Google Scholar 
Ter Steege, H. et al. Continental-scale patterns of canopy tree composition and function across Amazonia. Nature 443, 444–447 (2006).Article 
ADS 

Google Scholar 
Hedin, L. O., Brookshire, E. N. J., Menge, D. N. L. & Barron, A. R. The nitrogen paradox in tropical forest ecosystems. Annu. Rev. Ecol. Evol. Syst. 40, 613–635 (2009).Article 

Google Scholar 
Menge, D. N. L. et al. Patterns of nitrogen-fixing tree abundance in forests across Asia and America. J. Ecol. 107, 2598–2610 (2019).Article 
CAS 

Google Scholar 
Matson, W. J.Jr Herbivory in relation to plant nitrogen content. Annu. Rev. Ecol. Syst. 11, 119–161 (1980).Article 

Google Scholar 
Coley, P. D., Bateman, M. L. & Kusar, T. A. The effects of plant quality on caterpillar growth and defense against natural enemies. Oikos 115, 219–228 (2006).Article 

Google Scholar 
Wieder, W. R., Cleveland, C. C., Smith, W. K. & Todd-Brown, K. Future productivity and carbon storage limited by terrestrial nutrient availability. Nat. Geosci. 8, 441–444 (2015).Article 
ADS 
CAS 

Google Scholar 
Barron, A. R., Purves, D. W. & Hedin, L. O. Facultative nitrogen fixation by canopy legumes in a lowland tropical forest. Oecologia 165, 511–520 (2011).Article 
ADS 

Google Scholar 
McCulloch, L. A. & Porder, S. Light fuels while nitrogen suppresses symbiotic nitrogen fixation hotspots in neotropical canopy gap seedlings. New Phytol. 231, 1734–1745 (2021).Article 
CAS 

Google Scholar 
Brookshire, E. N. J. et al. Symbiotic N fixation is sufficient to support net aboveground biomass accumulation in a humid tropical forest. Sci Rep. 9, 7571 (2019).Article 
ADS 
CAS 

Google Scholar 
Gei, M. et al. Legume abundance along successional and rainfall gradients in Neotropical forests. Nat. Ecol. Evol. 2, 1104–1111 (2018).Article 

Google Scholar 
Vance, C. P. in Nitrogen-fixing Leguminous Symbioses. Nitrogen Fixation: Origins, Applications, and Research Progress, Vol. 7 (eds Dilworth, M. J. et al.) (Springer, 2008).Vitousek, P. M. & Howarth, R. W. Nitrogen limitation on land and in the sea: how can it occur? Biogeochemistry 13, 87–115 (1991).Article 

Google Scholar 
Menge, D. N. L., Levin, S. A. & Hedin, L. O. Evolutionary tradeoffs can select against nitrogen fixation and thereby maintain nitrogen limitation. Proc. Natl Acad. Sci. USA 105, 1573–1578 (2008).Article 
ADS 
CAS 

Google Scholar 
Sheffer, E., Batterman, S. A., Levin, S. A. & Hedin, L. O. Biome-scale nitrogen fixation strategies selected by climatic constraints on nitrogen cycle. Nat. Plants 1, 15182 (2015).Article 
CAS 

Google Scholar 
Vitousek, P. M. & Field, C. B. Ecosystem constraints to symbiotic nitrogen fixers: a simple model and its implications. Biogeochemistry 46, 179–202 (1999).Article 
CAS 

Google Scholar 
Coley, P. D. & Barone, J. A. Herbivory and plant defenses in tropical forests. Annu. Rev. Ecol. Syst. 27, 305–335 (1996).Article 

Google Scholar 
Fyllas, N. M. et al. Basin-wide variations in foliar properties of Amazonian forest: phylogeny, soils and climate. Biogeosciences 6, 2677–2708 (2009).Article 
ADS 

Google Scholar 
Batterman, S. A. et al. Phosphatase activity and nitrogen fixation reflect species differences, not nutrient trading or nutrient balance, across tropical rainforest trees. Ecol. Lett. 21, 1486–1495 (2018).Article 

Google Scholar 
Menge, D. N. L., Wolf, A. A. & Funk, J. L. Diversity of nitrogen fixation strategies in Mediterranean legumes. Nat. Plants 1, 15064 (2015).Article 
CAS 

Google Scholar 
Ritchie, M. E. & Tilman, D. Responses of legumes to herbivores and nutrients during succession on a nitrogen-poor soil. Ecol. Soc. Am. 76, 2648–2655 (1995).
Google Scholar 
Taylor, B. N. & Ostrowsky, L. R. Nitrogen-fixing and non-fixing trees differ in leaf chemistry and defence but not herbivory in a lowland Costa Rican rain forest. J. Trop. Ecol. 35, 270–279 (2019).Article 

Google Scholar 
Endara, M.-J. et al. Coevolutionary arms race versus host defense chase in a tropical herbivore–plant system. Proc. Natl Acad. Sci. USA 114, E7499–E7505 (2017).Article 
CAS 

Google Scholar 
Kursar, T. A. & Coley, P. D. Convergence in defense syndromes of young leaves in tropical rainforests. Biochem. Syst. Ecol. 31, 929–949 (2003).Article 
CAS 

Google Scholar 
Kursar, T. A. et al. The evolution of antiherbivore defenses and their contribution to species coexistence in the tropical tree genus Inga. Proc. Natl Acad. Sci. USA 106, 18073–18078 (2009).Article 
ADS 
CAS 

Google Scholar 
Taylor, B. N. & Menge, D. N. L. Light regulates tropical symbiotic nitrogen fixation more strongly than soil nitrogen. Nat. Plants 4, 655–661 (2018).Article 
CAS 

Google Scholar 
Adams, M., Turnbull, T., Sprent, J. & Buchmann, N. Legumes are different: leaf nitrogen, photosynthesis, and water use efficiency. Proc. Natl Acad. Sci. USA 113, 4098–4103 (2016).Article 
ADS 
CAS 

Google Scholar 
Coley, P. D. Effects of plant growth rate and leaf lifetime on the amount and type of anti-herbivore defense. Oecologia 74, 531–536 (1988).Article 
ADS 
CAS 

Google Scholar 
Batterman, S. A., Wurzburger, N. & Hedin, L. O. Nitrogen and phosphorus interact to control tropical symbiotic N2 fixation: a test in Inga punctata. J. Ecol. 101, 1400–1408 (2013).Article 
CAS 

Google Scholar 
Eichhorn, M. P., Nilus, R., Compton, S. G., Hartley, S. E. & Burslem, D. F. R. P. Herbivory of tropical rain forest tree seedlings correlates with future mortality. Ecology 91, 1092–1101 (2010).Article 

Google Scholar 
Wink, M. Evolution of secondary metabolites in legumes (Fabaceae). South African J. Bot. 89, 164–175 (2013).Article 
CAS 

Google Scholar 
Currano, E. D. & Jacobs, B. F. Bug-bitten leaves from the early Miocene of Ethiopia elucidate the impacts of plant nutrient concentrations and climate on insect herbivore communities. Glob. Planet. Change 207, 103655 (2021).Article 

Google Scholar 
Wieder, W. R., Cleveland, C. C., Lawrence, D. M. & Bonan, G. B. Effects of model structural uncertainty on carbon cycle projections: biological nitrogen fixation as a case study. Environ. Res. Lett. 10, 044016 (2015).Article 
ADS 

Google Scholar 
Sprent, J. I. Legume Nodulation: A Global Perspective (John Wiley, 2009).Leigh, E. G. Jr Tropical Forest Ecology: A View from Barro Colorado Island (Oxford Univ. Press, 1999).Comita, L. S., Muller-Landau, H. C., Aguilar, S. & Hubbell, S. P. Asymmetric density dependence shapes species abundances in a tropical tree community. Science 329, 330–332 (2010).Article 
ADS 
CAS 

Google Scholar 
Queenborough, S. A., Metz, M. R., Valencia, R. & Wright, S. J. Demographic consequences of chromatic leaf defence in tropical tree communities: do red young leaves increase growth and survival? Ann. Bot. 112, 677–684 (2013).Article 

Google Scholar 
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671 (2012).Article 
CAS 

Google Scholar 
Pasquini, S. C. & Santiago, L. S. Nutrients limit photosynthesis in seedlings of a lowland tropical forest tree species. Oecologia 168, 311–319 (2012).Article 
ADS 
CAS 

Google Scholar 
Collalti, A. & Prentice, I. C. Is NPP proportional to GPP? Waring’s hypothesis 20 years on. Tree Physiol. 39, 1473–1483 (2019).Article 
CAS 

Google Scholar 
Westbrook, J. W. et al. What makes a leaf tough? Patterns of correlated evolution between leaf toughness traits and demographic rates among 197 shade-tolerant woody species in a Neotropical forest. Am. Nat. 177, 800–811 (2011).Article 

Google Scholar 
Wright, S. J. et al. Functional traits and the growth–mortality trade‐off in tropical trees. Ecology 91, 3664–3674 (2010).Article 

Google Scholar 
Kitajima, K. et al. How cellulose-based leaf toughness and lamina density contribute to long leaf lifespans of shade-tolerant species. New Phytol. 195, 640–652 (2012).Article 

Google Scholar 
Kitajima, K., Wright, S. J. & Westbrook, J. W. Leaf cellulose density as the key determinant of inter- and intra-specific variation in leaf fracture toughness in a species-rich tropical forest. Interface Focus https://doi.org/10.1098/rsfs.2015.0100 (2016).Sedio, B. E., Echeverri, J. C. R., Boya, C. A. & Wright, S. J. Sources of variation in foliar secondary chemistry in a tropical forest tree community. Ecology 98, 616–623 (2017).Article 

Google Scholar 
Brooks, M. E. et al. glmmTMB balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling. R J. 9, 378–400 (2017).Article 

Google Scholar 
Smithson, M. & Verkuilen, J. A better lemon squeezer? Maximum-likelihood regression with beta-distributed dependent variables. Psychol. Methods 11, 54–71 (2006).Article 

Google Scholar 
Murphy, S. J., Xu, K. & Comita, L. S. Tree seedling richness, but not neighborhood composition, influences insect herbivory in a temperate deciduous forest community. Ecol. Evol. 6, 6310–6319 (2016).Article 

Google Scholar 
Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. Preprint at https://arxiv.org/abs/1406.5823 (2014).Moles, A. T. & Westoby, M. Do small leaves expand faster than large leaves, and do shorter expansion times reduce herbivore damage? Oikos 90, 517–524 (2000).Article 

Google Scholar 
Bürkner, P. C. brms: An R package for Bayesian multilevel models using Stan. J. Stat. Softw. https://doi.org/10.18637/jss.v080.i01 (2017). More

SamplingSediment samples were obtained from the Kap København Formation in North Greenland (82° 24′ 00″ N 22° 12′ 00″ W) in the summers of 2006, 2012 and 2016 (see Supplementary Table 3.1.1). Sampled material consisted of organic-rich permafrost and dry permafrost. Prior to sampling, profiles were cleaned to expose fresh material. Samples were hereafter collected vertically from the slope of the hills either using a 10 cm diameter diamond headed drill bit or cutting out ~40 × 40 × 40 cm blocks. Sediments were kept frozen in the field and during transportation to the lab facility in Copenhagen. Disposable gloves and scalpels were used and changed between each sample to avoid cross-contamination. In a controlled laboratory environment, the cores and blocks were further sub-sampled for material taking only the inner part of sediment cores, leaving 1.5–2 cm between the inner core and the surface that provided a subsample of approximately 6–10 g. Subsequently, all samples were stored at temperatures below −22 °C.We sampled organic-rich sediment by taking samples and biological replicates across the three stratigraphic units B1, B2 and B3, spanning 5 different sites, site: 50 (B3), 69 (B2), 74a (B1), 74b (B1) and 119 (B3). Each biological replicate from each unit at each site was further sampled in different sublayers (numbered L0–L4, Source Data 1, sheet 1).Absolute age datingIn 2014, Be and Al oxide targets from 8× 1 kg quartz-rich sand samples collected at modern depths ranging from 3 to 21 m below stream cut terraces were analysed by accelerator mass spectrometry and the cosmogenic isotope concentrations interpreted as maximum ages using a simple burial dating approach1 (26Al:10Be versus normalized 10Be). The 26Al and 10Be isotopes were produced by cosmic ray interactions with exposed quartz in regolith and bedrock surfaces in the mountains above Kap København prior to deposition. We assume that the 26Al:10Be was uniform and steady for long time periods in the upper few metres of these gradually eroding palaeo-surfaces. Once eroded by streams and hillslope processes, the quartz sand was deposited in sandy braided stream sediment, deltaic distributary systems, or the near-shore environment and remained effectively shielded from cosmic ray nucleons buried (many tens of metres) under sediment, intermittent ice shelf or ice sheet cover, and—at least during interglacials—the marine water column until final emergence. The simple burial dating approach assumes that the sand grains experienced only one burial event. If multiple burial events separated by periods of re-exposure occurred, then the starting 26Al:10Be before the last burial event would be less than the initial production ratio (6.75 to 7.42, see discussion below) owing to the relatively faster decay of 26Al during burial, and therefore the calculated burial age would be a maximum limiting age. Multiple burial events can be caused by shielding by thick glacier ice in the source area, or by sediment storage in the catchment prior to final deposition. These shielding events mean that the 26Al:10Be is lower, and therefore a calculated burial age assuming the initial production ratio would overestimate the final burial duration. We also consider that once buried, the sand grains may have been exposed to secondary cosmogenic muons (their depth would be too great for submarine nucleonic production). As sedimentation rates in these glaciated near-shore environments are relatively rapid, we show that even the muonic production would be negligible (see Supplemental Information). However, once the marine sediments emerged above sea level, in-situ production by both nucleogenic and muogenic production could alter the 26Al:10Be. The 26Al versus 10Be isochron plot reveals this complex burial history (Supplementary Information, section 3) and the concentration versus depth composite profiles for both 26Al and 10Be reveal that the shallowest samples may have been exposed during a period of time (~15,000 years ago) that is consistent with deglaciation in the area (Supplemental Information). While we interpret the individual simple burial age of all samples as a maximum limiting age of deposition of the Kap København Formation Member B, we recommend using the three most deeply shielded samples in a single depth profile to minimize the effect of post-depositional production. We then calculate a convolved probability distribution age for these three samples (KK06A, B and C). However, this calculation depends on the 26Al:10Be production ratio we use (that is, between 6.75 and 7.42) and on whether we adjust for erosion in the catchment. So, we repeat the convolved probability distribution function age for the lowest and highest production ratio and zero to maximum possible erosion rate, to obtain the minimum and maximum limiting age range at 1σ confidence (Supplementary Information, section 3). Taking the midpoint between the negative and positive 3σ confidence limits, we obtain a maximum burial age of 2.70 ± 0.46 Myr. This age is also supported by the position of those three samples on the isochron plot, which suggests the true age may not be significantly different that this maximum limiting age.Thermal ageThe extent of thermal degradation of the Kap København DNA was compared to the DNA from the Krestovka Mammoth molar. Published kinetic parameters for DNA degradation64 were used to calculate the relative rate difference over a given interval of the long-term temperature record and to quantify the offset from the reference temperature of 10 °C, thus estimating the thermal age in years at 10 °C for each sample (Supplementary Information, section 4). The mean annual air temperature (MAT) for the the Kap København sediment was taken from Funder et al. (2001)6 and for the Krestovka Mammoth the MAT was calculated using temperature data from the Cerskij Weather Station (WMO no. 251230) 68.80° N 161.28° E, 32 m from the International Research Institute Data Library (https://iri.columbia.edu/) (Supplementary Table 4.4.1).We did not correct for seasonal fluctuation for the thermal age calculation of the Kap København sediments or from the Krestovka Mammoth. We do provide theoretical average fragment length for four different thermal scenarios for the DNA in the Kap København sediments (Supplementary Table 4.4.2). A correction in the thermal age calculation was applied for altitude using the environmental lapse rate (6.49 °C km−1). We scaled the long-term temperature model of Hansen et al. (2013)65 to local estimates of current MATs by a scaling factor sufficient to account for the estimates of the local temperature decline at the last glacial maximum and then estimated the integrated rate using an activation energy (Ea) of 127 kJ mol−1 (ref. 64).Mineralogic compositionThe minerals in each of the Kap København sediment samples were identified using X-ray diffraction and their proportions were quantified using Rietveld refinement. The samples were homogenized by grinding ~1 g of sediment with ethanol for 10 min in a McCrone Mill. The samples were dried at 60 °C and added corundum (CR-1, Baikowski) as the internal standard to a final concentration of 20.0 wt%. Diffractograms were collected using a Bruker D8 Advance (Θ–Θ geometry) and the LynxEye detector (opening 2.71°), with Cu Kα1,2 radiation (1.54 Å; 40 kV, 40 mA) using a Ni-filter with thickness of 0.2 mm on the diffracted beam and a beam knife set at 3 mm. We scanned from 5–90° 2θ with a step size of 0.1° and a step time of 4 s while the sample was spun at 20 rpm. The opening of the divergence slit was 0.3° and of the antiscatter slit 3°. Primary and secondary Soller slits had an opening of 2.5° and the opening of the detector window was 2.71°. For the Rietveld analysis, we used the Profex interface for the BGMN software66,67. The instrumental parameters and peak broadening were determined by the fundamental parameters ray-tracing procedure68. A detailed description of identification of clay minerals can be found in the supporting information.AdsorptionWe used pure or purified minerals for adsorption studies. The minerals used and treatments for purifying them are listed in Supplementary Table 4.2.6. The purity of minerals was checked using X-ray diffraction with the same instrumental parameters and procedures as listed in the above section i.e., mineralogical composition. Notes on the origin, purification and impurities can be found in the Supplementary Information section 4. We used artificial seawater69 and salmon sperm DNA (low molecular weight, lyophilized powder, Sigma Aldrich) as a model for eDNA adsorption. A known amount of mineral powder was mixed with seawater and sonicated in an ultrasonic bath for 15 min. The DNA stock was then added to the suspension to reach a final concentration between 20–800 μg ml−1. The suspensions were equilibrated on a rotary shaker for 4 h. The samples were then centrifuged and the DNA concentration in the supernatant determined with UV spectrometry (Biophotometer, Eppendorf), with both positive and negative controls. All measurements were done in triplicates, and we made five to eight DNA concentrations per mineral. We used Langmuir and Freundlich equations to fit the model to the experimental isotherm and to obtain adsorption capacity of a mineral at a given equilibrium concentration.PollenThe pollen samples were extracted using the modified Grischuk protocol adopted in the Geological Institute of the Russian Academy of Science which utilizes sodium pyrophosphate and hydrofluoric acid70. Slides prepared from 6 samples were scanned at 400× magnification with a Motic BA 400 compound microscope and photographed using a Moticam 2300 camera. Pollen percentages were calculated as a proportion of the total palynomorphs including the unidentified grains. Only 4 of the 6 samples yielded terrestrial pollen counts ≥50. In these, the total palynomorphs identified ranged from 225 to 71 (mean = 170.25; median = 192.5). Identifications were made using several published keys71,72. The pollen diagram was initially compiled using Tilia version 1.5.1273 but replotted for this study using Psimpoll 4.1074.DNA recoveryFor recovery calculation, we saturated mineral surfaces with DNA. For this, we used the same protocol as for the determination of adsorption isotherms with an added step to remove DNA not adsorbed but only trapped in the interstitial pores of wet paste. This step was important because interstitial DNA would increase the amount of apparently adsorbed DNA and overestimate the recovery. To remove trapped DNA after adsorption, we redispersed the minerals in seawater. The process of redispersing the wet paste in seawater, ultracentrifugation and removal of supernatant lasted less than 2.5 min. After the second centrifugation, the wet pastes were kept frozen until extraction. We used the same extraction protocol as for the Kap København sediments. After the extraction, the DNA concentration was again determined using UV spectrometry.MetagenomesA total of 41 samples were extracted for DNA75 and converted to 65 dual-indexed Illumina sequencing libraries (including 13 negative extraction- and library controls)30. 34 libraries were thereafter subjected to ddPCR using a QX200 AutoDG Droplet Digital PCR System (Bio-Rad) following manufacturer’s protocol. Assays for ddPCR include a P7 index primer (5′-AGCAGAAGACGGCATAC-3′) (900nM), gene-targeting primer (900 nM), and a gene-targeting probe (250nM). We screened for Viridiplantae psbD (primer: 5′-TCATAATTGGACGTTGAACC-3′, probe: 5′-(FAM)ACTCCCATCATATGAAA(BHQ1)-3′) and Poaceae psbA (primer: 5′-CTCACAACTTCCCTCTAGAC-3′, probe 5′-(HEX)AGCTGCTGTTGAAGTTC(BHQ1)-3′). Additionally, 34 of the 65 libraries were enriched using targeted capture enrichment, for mammalian mitochondrial DNA using the PaleoChip Arctic1.0 bait-set31 and all libraries were hereafter sequenced on an Illumina HiSeq 4000 80 bp PE or a NovaSeq 6000 100 bp PE. We sequenced a total of 16,882,114,068 reads which, after low complexity filtering (Dust = 1), quality trimming (q ≥ 25), duplicate removal and filtering for reads longer than 29 bp (only paired read mates for NovaSeq data) resulted in 2,873,998,429 reads that were parsed for further downstream analysis. We next estimated kmer similarity between all samples using simka32 (setting heuristic count for max number of reads (-max-reads 0) and a kmer size of 31 (-kmer-size 31)), and performed a principal component analysis (PCA) on the obtained distance matrix (see Supplementary Information, ‘DNA’). We hereafter parsed all QC reads through HOLI33 for taxonomic assignment. To increase resolution and sensitivity of our taxonomic assignment, we supplemented the RefSeq (92 excluding bacteria) and the nucleotide database (NCBI) with a recently published Arctic-boreal plant database (PhyloNorway) and Arctic animal database34 as well as searched the NCBI SRA for 139 genomes of boreal animal taxa (March 2020) of which 16 partial-full genomes were found and added (Source Data 1, sheet 4) and used the GTDB microbial database version 95 as decoy. All alignments were hereafter merged using samtools and sorted using gz-sort (v. 1). Cytosine deamination frequencies were then estimated using the newly developed metaDMG, by first finding the lowest common ancestor across all possible alignments for each read and then calculating damage patterns for each taxonomic level36 (Supplementary Information, section 6). In parallel, we computed the mean read length as well as number of reads per taxonomic node (Supplementary Information, section 6). Our analysis of the DNA damage across all taxonomic levels pointed to a minimum filter for all samples at all taxonomic levels with a D-max ≥ 25% and a likelihood ratio (λ-LR) ≥ 1.5. This ensured that only taxa showing ancient DNA characteristics were parsed for downstream profiling and analysis and resulted in no taxa within any controls being found (Supplementary Information, section 6).Marine eukaryotic metagenomeWe sought to identify marine eukaryotes by first taxonomically labelling all quality-controlled reads as Eukaryota, Archaea, Bacteria or Virus using Kraken 276 with the parameters ‘–confidence 0.5 –minimum-hit-groups 3’ combined with an extra filtering step that only kept those reads with root-to-leaf score >0.25. For the initial Kraken 2 search, we used a coarse database created by the taxdb-integration workflow (https://github.com/aMG-tk/taxdb-integration) covering all domains of life and including a genomic database of marine planktonic eukaryotes63 that contain 683 metagenome-assembled genomes (MAGs) and 30 single-cell genomes (SAGs) from Tara Oceans77, following the naming convention in Delmont et al.63, we will refer to them as SMAGs. Reads labelled as root, unclassified, archaea, bacteria and virus were refined through a second Kraken 2 labelling step using a high-resolution database containing archaea, bacteria and virus created by the taxdb-integration workflow. We used the same Kraken 2 parameters and filtering thresholds as the initial search. Both Kraken 2 databases were built with parameters optimized for the study read length (–kmer-len 25 –minimizer-len 23 –minimizer-spaces 4).Reads labelled as eukaryota, root and unclassified were hereafter mapped with Bowtie278 against the SMAGs. We used MarkDuplicates from Picard (https://github.com/broadinstitute/picard) to remove duplicates and then we calculated the mapping statistics for each SMAG in the BAM files with the filterBAM program (https://github.com/aMG-tk/bam-filter). We furthermore estimated the postmortem damage of the filtered BAM files with the Bayesian methods in metaDMG and selected those SMAGs with a D-max ≥ 0.25 and a fit quality (λ-LR) higher than 1.5. The SMAGs with fewer than 500 reads mapped, a mean read average nucleotide identity (ANI) of less than than 93% and a breadth of coverage ratio and coverage evenness of less than 0.75 were removed. We followed a data-driven approach to select the mean read ANI threshold, where we explored the variation of mapped reads as a function of the mean read ANI values from 90% to 100% and identified the elbow point in the curve (Supplementary Fig. 6.11.1). We used anvi’o79 in manual mode to plot the mapping and damage results using the SMAGs phylogenomic tree inferred by Delmont et al. as reference. We used the oceanic signal of Delmont et al. as a proxy to the contemporary distribution of the SMAGs in each ocean and sea (Fig. 5 and Supplementary Information, section 6).Comparison of DNA, macrofossil and pollenTo allow comparison between records in DNA, macrofossil and pollen, the taxonomy was harmonized following the Pan Arctic Flora checklist43 and NCBI. For example, since Bennike (1990)18, Potamogeton has been split into Potamogeton and Stuckenia, Polygonym has been split to Polygonum and Bistorta, and Saxifraga was split to Saxifraga and Micranthes, whereas others have been merged, such as Melandrium with Silene40. Plant families have changed names—for instance, Gramineae is now called Poaceae and Scrophulariaceae has been re-circumscribed to exclude Plantaginaceae and Orobancheae80. We then classified the taxa into the following: category 1 all identical genus recorded by DNA and macrofossils or pollen, category 2 genera recorded by DNA also found by macrofossils or pollen including genus contained within family level classifications, category 3 taxa only recorded by DNA, category 4 taxa only recorded by macrofossils or pollen (Source Data 1).Phylogenetic placementWe sought to phylogenetically place the set of ancient taxa with the most abundant number of reads assigned, and with a sufficient number of reference sequences to build a phylogeny. These taxa include reads mapped to the chloroplast genomes of the flora genera Salix, Populus and Betula, and to the mitochondrial genomes of the fauna families Elephantidae, Cricetidae, Leporidae, as well as the subfamilies Capreolinae and Anserinae. Although the evolution of the chloroplast genome is somewhat less stable than that of the plant mitochondrial genome, it has a faster rate of evolution, and is non-recombining, and hence is more likely to contain more informative sites for our analysis than the plant mitochondria81. Like the mitochondrial genome, the chloroplast genome also has a high copy number, so that we would expect a high number of sedimentary reads mapping to it.For each of these taxa, we downloaded a representative set of either whole chloroplast or whole mitochondrial genome fasta sequences from NCBI Genbank82, including a single representative sequence from a recently diverged outgroup. For the Betula genus, we also included three chloroplast genomes from the PhyloNorway database34,83. We changed all ambiguous bases in the fasta files to N. We used MAFFT84 to align each of these sets of reference sequences, and inspected multiple sequence alignments in NCBI MSAViewer to confirm quality85. We trimmed mitochondrial alignments with insufficient quality due to highly variable control regions for Leporidae, Cricetidae and Anserinae by removing the d-loop in MegaX86.The BEAST suite49 was used with default parameters to create ultrametric phylogenetic trees for each of the five sets of taxa from the multiple sequence alignments (MSAs) of reference sequences, which were converted from Nexus to Newick format in Figtree (https://github.com/rambaut/figtree). We then passed the multiple sequence alignments to the Python module AlignIO from BioPython87 to create a reference consensus fasta sequence for each set of taxa. Furthermore, we used SNPSites88 to create a vcf file from each of the MSAs. Since SNPSites outputs a slightly different format for missing data than needed for downstream analysis, we used a custom R script to modify the vcf format appropriately. We also filtered out non-biallelic SNPs.From the damage filtered ngsLCA output, we extracted all readIDs uniquely classified to reference sequences within these respective taxa or assigned to any common ancestor inside the taxonomic group and converted these back to fastq files using seqtk (https://github.com/lh3/seqtk). We merged reads from all sites and layers to create a single read set for each respective taxon. Next, since these extracted reads were mapped against a reference database including multiple sequences from each taxon, the output files were not on the same coordinate system. To circumvent this issue and avoid mapping bias, we re-mapped each read set to the consensus sequence generated above for that taxon using bwa89 with ancient DNA parameters (bwa aln -n 0.001). We converted these reads to bam files, removed unmapped reads, and filtered for mapping quality  > 25 using samtools90. This produced 103,042, 39,306, 91,272, 182 and 129 reads for Salix, Populus, Betula, Elephantidae and Capreolinae, respectively.We next used pathPhynder62, a phylogenetic placement algorithm that identifies informative markers on a phylogeny from a reference panel, evaluates SNPs in the ancient sample overlapping these markers, and traverses the tree to place the ancient sample according to its derived and ancestral SNPs on each branch. We used the transversions-only filter to avoid errors due to deamination, except for Betula, Salix and Populus in which we used no filter due to sufficiently high coverage. Last, we investigated the pathPhynder output in each taxon set to determine the phylogenetic placement of our ancient samples (see Supplementary Information for discussion on phylogenetic placement).Based on the analysis described above we further investigated the phylogenetic placement within the genus Mammut, or mastodons. To avoid mapping reference biases in the downstream results, we first built a consensus sequence from all comparative mitochondrial genomes used in said analysis and mapped the reads identified in ngsLCA as Elephantidae to the consensus sequence. Consensus sequences were constructed by first aligning all sequences of interest using MAFFT84 and taking a majority rule consensus base in Geneious v2020.0.5 (https://www.geneious.com). We performed three analyses for phylogenetic placement of our sequence: (1) Comparison against a single representative from each Elephantidae species including the sea cow (Dugong dugon) as outgroup, (2) Comparison against a single representative from each Elephantidae species, and (3) Comparison against all published mastodon mitochondrial genomes including the Asian elephant as outgroup.For each of these analyses we first built a new reference tree using BEAST v1.10.4 (ref. 47) and repeated the previously described pathPhynder steps, with the exception that the pathPhynder tree path analysis for the Mammut SNPs was based on transitions and transversions, not restricting to only transversions due to low coverage.
Mammut americanum
We confirmed the phylogenetic placement of our sequence using a selection of Elephantidae mitochondrial reference sequences, GTR+G, strict clock, a birth-death substitution model, and ran the MCMC chain for 20,000,000 runs, sampling every 20,000 steps. Convergence was assessed using Tracer91 v1.7.2 and an effective sample size (ESS)  > 200. To determine the approximate age of our recovered mastodon mitogenome we performed a molecular dating analysis with BEAST47 v1.10.4. We used two separate approaches when dating our mastodon mitogenome, as demonstrated in a recent publication92. First, we determined the age of our sequence by comparing against a dataset of radiocarbon-dated specimens (n = 13) only. Secondly, we estimated the age of our sequence including both molecularly (n = 22) and radiocarbon-dated (n = 13) specimens using the molecular dates previously determined92. We utilized the same BEAST parameters as Karpinski et al.92 and set the age of our sample with a gamma distribution (5% quantile: 8.72 × 104, Median: 1.178 × 106; 95% quantile: 5.093 × 106; initial value: 74,900; shape: 1; scale: 1,700,000). In short, we specified a substitution model of GTR+G4, a strict clock, constant population size, and ran the Markov Chain Monte Carlo chain for 50,000,000 runs, sampling every 50,000 steps. Convergence of the run was again determined using Tracer.Molecular dating methodsIn this section, we describe molecular dating of the ancient birch (Betula) chloroplast genome using BEAST v1.10.4 (ref. 47). In principle, the genera Betula, Populus and Salix had both sufficiently high chloroplast genome coverage (with mean depth 24.16×, 57.06× and 27.04×, respectively, although this coverage is highly uneven across the chloroplast genome) and enough reference sequences to attempt molecular dating on these samples. Notably, this is one of the reasons we included a recently diverged outgroup with a divergence time estimate in each of these phylogenetic trees. However, our Populus sample clearly contained a mixture of different species, as seen from its inconsistent placement in the pathPhynder output. In particular, there were multiple supporting SNPs to both Populus balsamifera and Populus trichocarpa, and both supporting and conflicting SNPs on branches above. Furthermore, upon inspection, our Salix sample contained a surprisingly high number of private SNPs which is inconsistent with any ancient or even modern age, especially considering the number of SNPs assigned to the edges of the phylogenetic tree leading to other Salix sequences. We are unsure what causes this inconsistency but hypothesize that our Salix sample is also a mixed sample, containing multiple Salix species that diverged from the same placement branch on the phylogenetic tree at different time periods. This is supported by looking at all the reads that cover these private SNP sites, which generally appear to be from a mixed sample, with reads containing both alternate and reference alleles present at a high proportion in many cases. Alternatively, or potentially jointly in parallel, this could be a consequence of the high number of nuclear plastid DNA sequences (NUPTs) in Salix93. Because of this, we continued with only Betula.First, we downloaded 27 complete reference Betula chloroplast genome sequences and a single Alnus chloroplast genome sequence to use as an outgroup from the NCBI Genbank repository, and supplemented this with three Betula chloroplast sequences from the PhyloNorway database generated in a recent study29, for a total of 31 reference sequences. Since chloroplast sequences are circular, downloaded sequences may not always be in the same orientation or at the same starting point as is necessary for alignment, so we used custom code (https://github.com/miwipe/KapCopenhagen) that uses an anchor string to rotate the reference sequences to the same orientation and start them all from the same point. We created a MSA of these transformed reference sequences with Mafft84 and checked the quality of our alignment by eye in Seqotron94 and NCBI MsaViewer. Next, we called a consensus sequence from this MSA using the BioAlign consensus function87 in Python, which is a majority rule consensus caller. We will use this consensus sequence to map the ancient Betula reads to, both to avoid reference bias and to get the ancient Betula sample on the same coordinates as the reference MSA.From the last common ancestor output in metaDMG36, we extracted read sets for all units, sites and levels that were uniquely classified to the taxonomic level of Betula or lower, with at a minimum sequence similarity of 90% or higher to any Betula sequence, using Seqtk95. We mapped these read sets against the consensus Betula chloroplast genome using BWA89 with ancient DNA parameters (-o 2 -n 0.001 -t 20), then removed unmapped reads, quality filtered for read quality ≥25, and sorted the resulting bam files using samtools89. For the purpose of molecular dating, it is appropriate to consider these read sets as a single sample, and so we merged the resulting bam files into one sample using samtools. We used bcftools89 to make an mpileup and call a vcf file, using options for haploidy and disabling the default calling algorithm, which can slightly biases the calls towards the reference sequence, in favour of a majority call on bases that passed the default base quality cut-off of 13. We included the default option using base alignment qualities96, which we found greatly reduced the read depths of some bases and removed spurious SNPs around indel regions. Lastly, we filtered the vcf file to include only single nucleotide variants, because we do not believe other variants such as insertions or deletions in an ancient environmental sample of this type to be of sufficiently high confidence to include in molecular dating.We downloaded the gff3 annotation file for the longest Betula reference sequence, MG386368.1, from NCBI. Using custom R code97, we parsed this file and the associated fasta to label individual sites as protein-coding regions (in which we labelled the base with its position in the codon according to the phase and strand noted in the gff3 file), RNA, or neither coding nor RNA. We extracted the coding regions and checked in Seqotron94 and R that they translated to a protein alignment well (for example, no premature stop codons), both in the reference sequence and the associated positions in the ancient sequence. Though the modern reference sequence’s coding regions translated to a high-quality protein alignment, translating the associated positions in the ancient sequence with no depth cut-off leads to premature stop codons and an overall poor quality protein alignment. On the other hand, when using a depth cut-off of 20 and replacing sites in the ancient sequence which did not meet this filter with N, we see a high-quality protein alignment (except for the N sites). We also interrogated any positions in the ancient sequence which differed from the consensus, and found that any suspicious regions (for example, with multiple SNPs clustered closely together spatially in the genome) were removed with a depth cut-off of 20. Because of this, we moved forward only with sites in both the ancient and modern samples which met a depth cut-off of at least 20 in the ancient sample, which consisted of about 30% of the total sites.Next, we parsed this annotation through the multiple sequence alignment to create partitions for BEAST47. After checking how many polymorphic and total sites were in each, we decided to use four partitions: (1) sites belonging to protein-coding positions 1 and 2, (2) coding position 3, (3) RNA, or (4) non-coding and non-RNA. To ensure that these were high confidence sites, each partition also only included those positions which had at least depth 20 in the ancient sequence and had less than 3 total gaps in the multiple sequence alignment. This gave partitions which had 11,668, 5,828, 2,690 and 29,538 sites, respectively. We used these four partitions to run BEAST47 v1.10.4, with unlinked substitution models for each partition and a strict clock, with a different relative rate for each partition. (There was insufficient information in these data to infer between-lineage rate variation from a single calibration). We assigned an age of 0 to all of the reference sequences, and used a normal distribution prior with mean 61.1 Myr and standard deviation 1.633 Myr for the root height48; standard deviation was obtained by conservatively converting the 95% HPD to z-scores. For the overall tree prior, we selected the coalescent model. The age of the ancient sequence was estimated following the overall procedures of Shapiro et al. (2011)98. To assess sensitivity to prior choice for this unknown date, we used two different priors, namely a gamma distribution metric towards a younger age (shape = 1, scale = 1.7); and a uniform prior on the range (0, 10 Myr). We also compared two different models of rate variation among sites and substitution types within each partition, namely a GTR+G with four rate categories, and base frequencies estimated from the data, and the much simpler Jukes Cantor model, which assumed no variation between substitution types nor sites within each partition. All other priors were set at their defaults. Neither rate model nor prior choice had a qualitative effect on results (Extended Data Fig. 10). We also ran the coding regions alone, since they translated correctly and are therefore highly reliable sites and found that they gave the same median and a much larger confidence interval, as expected when using fewer sites (Extended Data Fig. 10). We ran each Markov chain Monte Carlo for a total of 100 million iterations. After removing a burn-in of the first 10%, we verified convergence in Tracer91 v1.7.2 (apparent stationarity of traces, and all parameters having an Effective Sample Size  > 100). We also verified that the resulting MCC tree from TreeAnnotator47 had placed the ancient sequence phylogenetically identically to pathPhynder62 placement, which is shown in Extended Data Fig. 9. For our major results, we report the uniform ancient age prior, and the GTR+G4 model applied to each of the four partitions. The associated XML is given in Source Data 3. The 95% HPD was (2.0172,0.6786) for the age of the ancient Betula chloroplast sequence, with a median estimate of 1.323 Myr, as shown in Fig. 2.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article. More

Keeling, P. J. & Burki, F. Progress towards the tree of eukaryotes. Curr. Biol. 29, R808–R817 (2019).Article 
CAS 

Google Scholar 
Gawryluk, R. M. R. et al. Non-photosynthetic predators are sister to red algae. Nature 572, 240–243 (2019).Article 
CAS 

Google Scholar 
Janouškovec, J. et al. A new lineage of eukaryotes illuminates early mitochondrial genome reduction. Curr. Biol. 27, 3717–3724 (2017).Article 

Google Scholar 
Lax, G. et al. Hemimastigophora is a novel supra-kingdom-level lineage of eukaryotes. Nature 564, 410–414 (2018).Article 
ADS 
CAS 

Google Scholar 
Oren, A. Prokaryote diversity and taxonomy: current status and future challenges. Philos. Trans. R. Soc. Lond. B 359, 623–638 (2004).Article 
CAS 

Google Scholar 
Shu, W. S. & Huang, L. N. Microbial diversity in extreme environments. Nat. Rev. Microbiol. 20, 219–235 (2022).Article 
CAS 

Google Scholar 
Massana, R., del Campo, J., Sieracki, M. E., Audic, S. & Logares, R. Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles. ISME J. 8, 854–866 (2014).Article 

Google Scholar 
de Vargas, C. et al. Eukaryotic plankton diversity in the sunlit ocean. Science 348, 1261605 (2015).Article 

Google Scholar 
Flegontova, O. et al. Extreme diversity of diplonemid eukaryotes in the ocean. Curr. Biol. 26, 3060–3065 (2016).Article 
CAS 

Google Scholar 
Ahlering, M. A. & Carrel, J. E. Predators are rare even when they are small. Oikos 95, 471–475 (2001).Article 

Google Scholar 
Hehenberger, E. et al. Novel predators reshape holozoan phylogeny and reveal the presence of a two-component signaling system in the ancestor of animals. Curr. Biol. 27, 2043–2050 (2017).Article 
CAS 

Google Scholar 
Tikhonenkov, D. V. et al. Description of Colponema vietnamica sp. n. and Acavomonas peruviana n. gen. n. sp., two new alveolate phyla (Colponemidia nom. nov. and Acavomonidia nom. nov.) and their contributions to reconstructing the ancestral state of alveolates and eukaryotes. PLoS ONE 9, e95467 (2014).Article 
ADS 

Google Scholar 
Tikhonenkov, D. V. et al. New lineage of microbial predators adds complexity to reconstructing the evolutionary origin of animals. Curr. Biol. 30, 4500–4509 (2020).Article 
CAS 

Google Scholar 
Mylnikov, A. P. & Tikhonenkov, D. V. The new alveolate carnivorous flagellate Colponema marisrubri sp. n. (Colponemida, Alveolata) from the Red Sea. Zool. Zh. 88, 1163–1169 (2009).
Google Scholar 
Strassert, J. F. H., Irisarri, I., Williams, T. A. & Burki, F. A molecular timescale for eukaryote evolution with implications for the origin of red algal-derived plastids. Nat. Commun. 12, 1879 (2021).Article 
ADS 
CAS 

Google Scholar 
Rodriguez-Ezpeleta, N. et al. Detecting and overcoming systematic errors in genome-scale phylogenies. Syst. Biol. 56, 389–399 (2007).Article 
CAS 

Google Scholar 
Strassert, J. F. H., Jamy, M., Mylnikov, A. P., Tikhonenkov, D. V. & Burki, F. New phylogenomic analysis of the enigmatic phylum Telonemia further resolves the eukaryote tree of life. Mol. Biol. Evol. 36, 757–765 (2019).Article 
CAS 

Google Scholar 
Lanfear, R., Kokko, H. & Eyre-Walker, A. Population size and the rate of evolution. Trends Ecol. Evol. 29, 33–41 (2014).Article 

Google Scholar 
Bahler, M. & Rhoads, A. Calmodulin signaling via the IQ motif. FEBS Lett. 513, 107–113 (2002).Article 
CAS 

Google Scholar 
Schaffer, D. E., Iyer, L. M., Burroughs, A. M. & Aravind, L. Functional innovation in the evolution of the calcium-dependent system of the eukaryotic endoplasmic reticulum. Front. Genet. 11, 34 (2020).Article 

Google Scholar 
Morita-Yamamuro, C. et al. The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain. Plant Cell Physiol. 46, 902–912 (2005).Article 
CAS 

Google Scholar 
Rosado, C. J. et al. The MACPF/CDC family of pore-forming toxins. Cell. Microbiol. 10, 1765–1774 (2008).Article 
CAS 

Google Scholar 
Ishino, T., Chinzei, Y. & Yuda, M. A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cell. Microbiol. 7, 199–208 (2005).Article 
CAS 

Google Scholar 
Satoh, H., Oshiro, N., Iwanaga, S., Namikoshi, M. & Nagai, H. Characterization of PsTX-60B, a new membrane-attack complex/perforin (MACPF) family toxin, from the venomous sea anemone Phyllodiscus semoni. Toxicon 49, 1208–1210 (2007).Article 
CAS 

Google Scholar 
Tikhonenkov, D. V., Mazei, Y. A. & Embulaeva, E. A. Degradation succession of heterotrophic flagellate communities in microcosms. Zh. Obs. Biol. 69, 57–64 (2008).CAS 

Google Scholar 
Tikhonenkov, D. V. et al. On the origin of TSAR: morphology, diversity and phylogeny of Telonemia. Open Biol. 12, 210325 (2022).Article 
CAS 

Google Scholar 
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).Article 
CAS 

Google Scholar 
Keeling, P. J., Poulson, N. & McFadden, G. I. Phylogenetic diversity of parabasalian symbionts from termites, including the phylogenetic position of Pseudotrypanosoma and Trichonympha. J. Eukaryot. Microbiol. 45, 643–650 (1998).Article 
CAS 

Google Scholar 
Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71, 491–499 (1988).Article 
CAS 

Google Scholar 
Tikhonenkov, D. V., Janouškovec, J., Keeling, P. J. & Mylnikov, A. P. The morphology, ultrastructure and SSU rRNA gene sequence of a new freshwater flagellate, Neobodo borokensis n. sp. (Kinetoplastea, Excavata). J. Eukaryot. Microbiol. 63, 220–232 (2016).Article 
CAS 

Google Scholar 
Andrews, S. FastQC: a quality control tool for high throughput sequence data (Babraham Bioinformatics, 2010); https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.Zhang, J., Kobert, K., Flouri, T. & Stamatakis, A. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30, 614–620 (2013).Article 

Google Scholar 
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).Article 
CAS 

Google Scholar 
Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011).Article 
CAS 

Google Scholar 
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).Article 
CAS 

Google Scholar 
Laetsch, D. R. & Blaxter, M. L. BlobTools: interrogation of genome assemblies. F1000Research 6, 1287 (2017).Article 

Google Scholar 
Haas, B. J. et al. Denovo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494–1512 (2013).Article 
CAS 

Google Scholar 
Li, W. & Godzik, A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658–1659 (2006).Article 
CAS 

Google Scholar 
Buchfink, B., Xie, C. & Huson, D. H. Fast and sensitive protein alignment using DIAMOND. Nat. Methods 12, 59–60 (2015).Article 
CAS 

Google Scholar 
Shen, W. & Ren, H. TaxonKit: a practical and efficient NCBI taxonomy toolkit. J. Genet. Genomics 48, 844–850 (2021).Richter, D. J. et al. EukProt: a database of genome-scale predicted proteins across the diversity of eukaryotes. Peer Community Journal 2, e56 (2022).Simao, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31, 3210–3212 (2015).Article 
CAS 

Google Scholar 
Kanehisa, M., Furumichi, M., Sato, Y., Ishiguro-Watanabe, M. & Tanabe, M. KEGG: integrating viruses and cellular organisms. Nucleic Acids Res. 49, D545–D551 (2021).Article 
CAS 

Google Scholar 
Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A. C. & Kanehisa, M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 35, W182–W185 (2007).Article 

Google Scholar 
Burki, F. The eukaryotic tree of life from a global phylogenomic perspective. Cold Spring Harb. Perspect. Biol. 6, a016147 (2014).Article 

Google Scholar 
Waskom, M. et al. mwaskom/Seaborn: v0.8.1 (September 2017). Zenodo https://doi.org/10.5281/zenodo.883859 (2017).Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).Article 
ADS 
MathSciNet 
CAS 

Google Scholar 
Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279–D285 (2016).Article 
CAS 

Google Scholar 
Letunic, I. & Bork, P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 46, D493–D496 (2018).Article 
CAS 

Google Scholar 
Almagro Armenteros, J. J. et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 37, 420–423 (2019).Article 
CAS 

Google Scholar 
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).Article 
CAS 

Google Scholar 
Burns, J. A., Pittis, A. A. & Kim, E. Gene-based predictive models of trophic modes suggest Asgard archaea are not phagocytotic. Nat. Ecol. Evol. 2, 697–704 (2018).Article 

Google Scholar 
Emms, D. M. & Kelly, S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 20, 238 (2019).Article 

Google Scholar 
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).Article 
CAS 

Google Scholar 
Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98 (1999).CAS 

Google Scholar 
Minh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534 (2020).Article 
CAS 

Google Scholar 
Whelan, S., Irisarri, I. & Burki, F. PREQUAL: detecting non-homologous characters in sets of unaligned homologous sequences. Bioinformatics 34, 3929–3930 (2018).CAS 

Google Scholar 
Capella-Gutierrez, S., Silla-Martinez, J. M. & Gabaldon, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).Article 
CAS 

Google Scholar 
Roure, B., Rodriguez-Ezpeleta, N. & Philippe, H. SCaFoS: a tool for selection, concatenation and fusion of sequences for phylogenomics. BMC Evol. Biol. 7, S2 (2007).Article 

Google Scholar 
Lartillot, N., Rodrigue, N., Stubbs, D. & Richer, J. PhyloBayes MPI: phylogenetic reconstruction with infinite mixtures of profiles in a parallel environment. Syst. Biol. 62, 611–615 (2013).Article 
CAS 

Google Scholar 
Dayhoff, M., Schwartz, R. & Orcutt, B. in Atlas of Protein Sequence and Structure (ed. Dayhoff, M.) 345–352 (National Biomedical Research Foundation, 1978).Susko, E. & Roger, A. J. On reduced amino acid alphabets for phylogenetic inference. Mol. Biol. Evol. 24, 2139–2150 (2007).Article 
CAS 

Google Scholar 
Lartillot, N. & Philippe, H. A Bayesian mixture model for across-site heterogeneities in the amino-acid replacement process. Mol. Biol. Evol. 21, 1095–1109 (2004).Article 
CAS 

Google Scholar 
Quang le, S., Gascuel, O. & Lartillot, N. Empirical profile mixture models for phylogenetic reconstruction. Bioinformatics 24, 2317–2323 (2008).Article 

Google Scholar 
Wang, H. C., Minh, B. Q., Susko, E. & Roger, A. J. Modeling site heterogeneity with posterior mean site frequency profiles accelerates accurate phylogenomic estimation. Syst. Biol. 67, 216–235 (2018).Article 
CAS 

Google Scholar 
Kück, P. & Struck, T. H. BaCoCa—a heuristic software tool for the parallel assessment of sequence biases in hundreds of gene and taxon partitions. Mol. Phylogenet. Evol. 70, 94–98 (2014).Article 

Google Scholar 
Shimodaira, H. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51, 492–508 (2002).Article 

Google Scholar 
Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).Article 
CAS 

Google Scholar 
Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).Article 
MathSciNet 
CAS 

Google Scholar 
Dierckxsens, N., Mardulyn, P. & Smits, G. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 45, e18 (2017).
Google Scholar 
Kuznetsov, A. & Bollin, C. J. in Multiple Sequence Alignment (ed. Katoh, K.) 261–295 (Springer, 2021).Lohse, M., Drechsel, O., Kahlau, S. & Bock, R. OrganellarGenomeDRAW—a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets. Nucleic Acids Res. 41, W575–W581 (2013).Article 

Google Scholar 
Johnson, P. Z., Kasprzak, W. K., Shapiro, B. A. & Simon, A. E. RNA2Drawer: geometrically strict drawing of nucleic acid structures with graphical structure editing and highlighting of complementary subsequences. RNA Biol. 16, 1667–1671 (2019).Article 

Google Scholar 
Burger, G., Gray, M. W., Forget, L. & Lang, B. F. Strikingly bacteria-like and gene-rich mitochondrial genomes throughout jakobid protists. Genome Biol. Evol. 5, 418–438 (2013).Article 

Google Scholar 
Criscuolo, A. & Gribaldo, S. BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol. Biol. 10, 210 (2010).Article 

Google Scholar 
Zhang, D. et al. PhyloSuite: an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol. Ecol. Resour. 20, 348–355 (2020).Article 

Google Scholar 
Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).Article 
CAS 

Google Scholar 
Ibarbalz, F. M. et al. Global trends in marine plankton diversity across kingdoms of life. Cell 179, 1084–1097 (2019).Article 
CAS 

Google Scholar 
Massana, R. et al. Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing. Environ. Microbiol. 17, 4035–4049 (2015).Article 
CAS 

Google Scholar 
Gendron, E. M. S., Darcy, J. L., Hell, K. & Schmidt, S. K. Structure of bacterial and eukaryote communities reflect in situ controls on community assembly in a high-alpine lake. J. Microbiol. 57, 852–864 (2019).Article 
CAS 

Google Scholar 
Minerovic, A. D. et al. 18S-V9 DNA metabarcoding detects the effect of water-quality impairment. Ecol. Indic. 113, 106225 (2020).Article 
CAS 

Google Scholar 
Pearman, J. K. et al. Cross-shelf investigation of coral reef cryptic benthic organisms reveals diversity patterns of the hidden majority. Sci. Rep. 8, 8090 (2018).Article 
ADS 
CAS 

Google Scholar 
Rodas, A. M. et al. Eukaryotic plankton communities across reef environments in Bocas del Toro Archipelago, Panamá. Coral Reefs 39, 1453–1467 (2020).Article 

Google Scholar 
Schoenle, A. et al. High and specific diversity of protists in the deep-sea basins dominated by diplonemids, kinetoplastids, ciliates and foraminiferans. Commun. Biol. 4, 501 (2021).Article 
CAS 

Google Scholar 
Schulhof, M. A. et al. Sierra Nevada mountain lake microbial communities are structured by temperature, resources and geographic location. Mol. Ecol. 29, 2080–2093 (2020).Article 
CAS 

Google Scholar 
Yi, Z. et al. High-throughput sequencing of microbial eukaryotes in Lake Baikal reveals ecologically differentiated communities and novel evolutionary radiations. FEMS Microbiol. Ecol. 93, fix073 (2017). More

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

Predictors of reservoir statusOur analyses include all known rodent reservoirs for zoonotic pathogens (282 species). These reservoirs harbour a total of 95 known zoonotic pathogens (34 viruses, 26 bacteria, 17 helminths, 12 protozoa and six fungi) employing all known modes of transmission (43 vector-borne, 32 close-contact, 28 non-close contact, and 13 using multiple transmission modes) (Supplementary Data 2). Compared to presumed non-reservoirs (species currently not known to harbour any zoonotic pathogens), we observed that reservoir rodents are strikingly synanthropic (Figs. 2, 3a, Table 1). Despite potential geographic biases, and the general possibility that synanthropic species are better studied compared to non-synanthropic species (see Sampling bias and Supplementary Figs. 1, 2), synanthropy emerged as a defining characteristic of nearly all (95%) currently known rodent reservoirs. Of the 155 synanthropic species, only six are considered as truly synanthropic, i.e., predominately, if not exclusively, occurring in or near human dwellings, while the remaining species only occasionally show synanthropic behaviour (Supplementary Data 1).Fig. 2: Predictors of reservoir status.Final structural equation model linking reservoir status of rodent species (n = 269) with their synanthropy and hunting status, population fluctuations (s-index, log-transformed), and adult body mass, controlling for their occurrence in a range of habitats and the number of studies available per species. One-sided (directional) arrows represent a causal influence originating from the variable at the base of the arrow, with the width of the arrow and associated value representing the standardised strength of the relationship. The small double-sided arrows and numbers next to each response (endogenous) variable represent the error variance.Full size imageFig. 3: Characteristics of reservoir and synanthropic rodents.a Reservoir rodents are predominately synanthropic (n = 436 with n (non-reservoir) = 154, n (reservoir) = 282). b Synanthropic rodents display high population fluctuations (high s-index) (n = 269) and c, occur in multiple artificial habitats (n = 269) (Tables 1–3). In a, estimated probability and 95% confidence intervals are shown and in b–c, estimated probability is shown and shaded areas show 95 % confidence intervals.Full size imageTable 1 Summary of best-fit generalized linear mixed effects model for reservoir status (n = 436)Full size tableCompared to non-reservoirs, we also found that rodent reservoirs are disproportionately exploited by humans (hunted for meat and fur). Seventy-two of the regularly hunted rodent species (n = 83) are reservoirs (87%), and hunted rodent species harbour on average five times the number of zoonotic pathogens than non-hunted species (Table 2).Table 2 Summary of rodent characteristics divided by rodent group with respect to hunting, reservoir status, and synanthropic behaviourFull size tableWe explored causal pathways using a structural equation model (SEM) linking synanthropy, reservoir status, and their hypothesized predictors. The final model, which we established a priori, had 17 free parameters and 21 degrees of freedom (n = 269). The model fit, based on the SRMR (standardized root mean squared residual) and the RMSEA (root mean squared error of approximation) indicated a good fit (see Methods). From the initially formulated full model, the pathways linking reservoir status to population fluctuations (s-index, Methods), occurrence in grasslands, number of artificial habitats a species occurs in, and number of studies found per species were not significant and thus removed from the final model (Supplementary Fig. 3). Similarly, pathways linking synanthropy and occurrence in grasslands were not significant and also removed. All reported coefficients for pathways are standardized to facilitate comparisons among the different relationships. The relationships and coefficients below all refer to those in the final model.The focal variable in the model was reservoir status, which was strongly and positively associated with synanthropy and had the highest estimated pathway coefficient (standardised estimate = 0.58, 95% CI 0.49–0.66, Fig. 2). Controlling for synanthropy, species were more likely to be a reservoir with increasing adult weight (0.13, 0.04–0.22). Species that occur in savanna were less likely to be reservoirs (−0.13, −0.22 to −0.04), while hunted species were more likely to be reservoirs (Fig. 2, 0.20, 0.11–0.30).Synanthropy was influenced by four habitat variables: a species was more likely to be synanthropic if it occurs in a higher number of artificial habitats (0.17, 0.04–0.31), and occurs in urban areas (0.14, 0.01–0.27), deserts (0.12, 0.01–0.23), or forests (0.13, 0.02–0.24). Notably, species with higher s-index, and thus larger population fluctuations, were more likely to be synanthropic (0.12, 0.01–0.22), and the s-index itself decreased as adult weight increased (−0.16, −0.27 to −0.04). Finally, hunted species were characterized by higher adult bodyweight (0.35, 0.25–0.44) (Fig. 2).The number of studies per species was positively associated with both a species’ synanthropic behaviour (0.29, 0.19–0.39) and its reservoir status (0.09, 0.00– 0.19), albeit with weaker evidence for the latter effect (p = 0.054) (Fig. 2),The confirmatory generalized linear mixed effects models (GLMMs) (Tables 1, 3), which control for correlation among species within the same family, showed that our SEM results were robust. Indeed, synanthropy was a significant predictor of reservoir status. These models underscore synanthropy as the most important predictor of reservoir status in our analysis (Table 1, Figs. 2–3).Table 3 Summary of best-fit generalized linear mixed effects model for synanthropic status (n = 269)Full size tablePopulation fluctuations affect transmission riskOur newly compiled data on the magnitude of population fluctuations enabled comparative investigations beyond theoretically straightforward predictions that transmission risk increases with reservoir abundance for density-dependent systems. We show that while strong population fluctuations (measured as the s-index) are found frequently in both reservoir and non-reservoir rodents (Table 2), synanthropic rodents exhibit much larger population fluctuations compared to non-synanthropic rodents (Table 2, Figs. 2–3). This pattern was apparent despite broad confidence intervals in the relationship between the s-index and the probability of being synanthropic (Fig. 3b, Tables 2, 3). Taken together, our results suggest that larger population fluctuations in reservoir species increase zoonotic transmission risk via synanthropic behaviours of rodents, thereby increasing the likelihood of zoonotic spillover infection to humans.Habitat generalism and habitat transformation increase transmission riskWe also find that reservoir species thrive in human-created (artificial) habitats (Fig. 3a, c, Tables 2–3), which reflects a general flexibility in their use of diverse habitat types compared to non-reservoir species (Fig. 4a, Table 2). In addition, the number of zoonotic pathogens harboured by a rodent species increased with habitat breadth (r436 = 0.34, p  More