Ecology

When I first came to the Amazon from central Brazil in 1978, I was planning to stay just a year, but I was mesmerized by the size of the rainforest’s rivers and its biodiversity. I ended up staying longer and earned my master’s degree in aquatic biology in 1984 from the National Institute for Amazonian Research (INPA), in Manaus, Brazil. I then went to get my PhD in ecology and evolutionary biology at the University of Arizona in Tucson, and returned to Manaus in 1998 to work as an ichthyologist at INPA.I was part of the team that started INPA’s fish collection in 1978. At the time, most scientific information on Amazonian fish, including specimens, had been collected by researchers and stored at other institutions around the world. Brazilians couldn’t easily access any of it. Now, INPA has preserved and catalogued more than 600,000 fish, all of which are available to our graduate students and scientific community.
Women in science
This picture, from last June, was taken at a Manicoré River creek in northwest Brazil during a Greenpeace expedition. I’m holding a bag of small fish, collected using sieves.Since 2006, the riverside communities on the Manicoré have been advocating for a reserve to protect their land from non-sustainable practices. They asked Greenpeace to help map the area’s biodiversity to bolster their application. Greenpeace in turn invited INPA researchers for its mapping expedition. We spent 20 days collecting and registering the wide range of creatures in the Manicoré’s basins.Besides fires, the Amazon has been hit hard by deforestation and industrial activities. We registered a decline in populations of several fish species after the construction of the hydroelectric complex of Belo Monte — the second- largest in the world — in the Xingu River. These species can thrive only in the oxygenated environment of running rivers and waterfalls, which have been largely destroyed. More

Ackerly, D. D. Community assembly, niche conservatism, and adaptive evolution in changing environments. Int. J. Plant Sci. 164, S165–S184 (2003).Article 

Google Scholar 
Kellermann, V., Van Heerwaarden, B., Sgrò, C. M. & Hoffmann, A. A. Fundamental evolutionary limits in ecological traits drive Drosophila species distributions. Science 325, 1244–1246 (2009).Article 
CAS 

Google Scholar 
Hansen, M. M., Olivieri, I., Waller, D. M. & Nielsen, E. E. Monitoring adaptive genetic responses to environmental change. Mol. Ecol. 21, 1311–1329 (2012).Article 

Google Scholar 
Aitken, S. N. & Whitlock, M. C. Assisted gene flow to facilitate local adaptation to climate change. Annu. Rev. Ecol. Evol. Syst. 44, 367–388 (2013).Article 

Google Scholar 
Becker, M. et al. Hybridization may facilitate in situ survival of endemic species through periods of climate change. Nat. Clim. Change 3, 1039–1043 (2013).Article 

Google Scholar 
Allendorf, F. W., Leary, R. F., Spruell, P. & Wenburg, J. K. The problems with hybrids: setting conservation guidelines. Trends Ecol. Evol. 16, 613–622 (2001).Article 

Google Scholar 
Todesco, M. et al. Hybridization and extinction. Evol. Appl. 9, 892–908 (2016).Article 
CAS 

Google Scholar 
Rhymer, J. M. & Simberloff, D. Extinction by hybridization and introgression. Annu. Rev. Ecol. Syst. 27, 83–109 (1996).Article 

Google Scholar 
Taylor, S. A. & Larson, E. L. Insights from genomes into the evolutionary importance and prevalence of hybridization in nature. Nat. Ecol. Evol. 3, 170–177 (2019).Article 

Google Scholar 
vonHoldt, B. M., Brzeski, K. E., Wilcove, D. S. & Rutledge, L. Y. Redefining the role of admixture and genomics in species conservation. Conserv. Lett. 11, e12371 (2018).Article 

Google Scholar 
Hamilton, J. A. & Miller, J. M. Adaptive introgression as a resource for management and genetic conservation in a changing climate. Conserv. Biol. 30, 33–41 (2016).Article 

Google Scholar 
Ralls, K., Sunnucks, P., Lacy, R. C. & Frankham, R. Genetic rescue: a critique of the evidence supports maximizing genetic diversity rather than minimizing the introduction of putatively harmful genetic variation. Biol. Conserv. 251, 108784 (2020).Article 

Google Scholar 
Capblancq, T., Fitzpatrick, M. C., Bay, R. A., Exposito-Alonso, M. & Keller, S. R. Genomic prediction of (mal) adaptation across current and future climatic landscapes. Annu. Rev. Ecol. Evol. Syst. 51, 245–269 (2020).Article 

Google Scholar 
Rellstab, C., Dauphin, B. & Exposito‐Alonso, M. Prospects and limitations of genomic offset in conservation management. Evol. Appl. 14, 1202–1212 (2021).Article 

Google Scholar 
Bay, R. A. et al. Genomic signals of selection predict climate-driven population declines in a migratory bird. Science 359, 83–86 (2018).Article 
CAS 

Google Scholar 
Rellstab, C. et al. Signatures of local adaptation in candidate genes of oaks (Quercus spp.) with respect to present and future climatic conditions. Mol. Ecol. 25, 5907–5924 (2016).Article 

Google Scholar 
Fitzpatrick, M. C. & Keller, S. R. Ecological genomics meets community-level modelling of biodiversity: mapping the genomic landscape of current and future environmental adaptation. Ecol. Lett. 18, 1–16 (2015).Article 

Google Scholar 
Exposito-Alonso, M. et al. Genomic basis and evolutionary potential for extreme drought adaptation in Arabidopsis thaliana. Nat. Ecol. Evol. 2, 352–358 (2018).Article 

Google Scholar 
Kindt, R. AlleleShift: an R package to predict and visualize population-level changes in allele frequencies in response to climate change. PeerJ 9, e11534 (2021).Article 

Google Scholar 
Gain, C. & François, O. LEA 3: factor models in population genetics and ecological genomics with R. Mol. Ecol. Resour. 21, 2738–2748 (2020).Article 

Google Scholar 
Aguirre-Liguori, J. A., Ramírez-Barahona, S. & Gaut, B. S. The evolutionary genomics of species’ responses to climate change. Nat. Ecol. Evol. 5, 1350–1360 (2021).Article 

Google Scholar 
Taylor, S. A., Larson, E. L. & Harrison, R. G. Hybrid zones: windows on climate change. Trends Ecol. Evol. 30, 398–406 (2015).Article 

Google Scholar 
Hoffmann, A. A. & Sgro, C. M. Climate change and evolutionary adaptation. Nature 470, 479–485 (2011).Article 
CAS 

Google Scholar 
McGuigan, K., Franklin, C. E., Moritz, C. & Blows, M. W. Adaptation of rainbow fish to lake and stream habitats. Evolution 57, 104–118 (2003).
Google Scholar 
Smith, S., Bernatchez, L. & Beheregaray, L. RNA-seq analysis reveals extensive transcriptional plasticity to temperature stress in a freshwater fish species. BMC Genomics 14, 375 (2013).Article 
CAS 

Google Scholar 
Smith, S. et al. Latitudinal variation in climate‐associated genes imperils range edge populations. Mol. Ecol. 29, 4337–4349 (2020).Article 
CAS 

Google Scholar 
Sandoval-Castillo, J. et al. Adaptation of plasticity to projected maximum temperatures and across climatically defined bioregions. Proc. Natl Acad. Sci. USA 117, 17112–17121 (2020).Article 
CAS 

Google Scholar 
Brauer, C., Unmack, P. J., Smith, S., Bernatchez, L. & Beheregaray, L. B. On the roles of landscape heterogeneity and environmental variation in determining population genomic structure in a dendritic system. Mol. Ecol. 27, 3484–3497 (2018).Article 
CAS 

Google Scholar 
Attard, C. R. et al. Fish out of water: genomic insights into persistence of rainbowfish populations in the desert. Evolution 76, 171–183 (2022).Article 

Google Scholar 
Gates, K. et al. Environmental selection, rather than neutral processes, best explain patterns of diversity in a tropical rainforest fish. Preprint at bioRxiv https://doi.org/10.1101/2022.1105.1113.491913 (2022).Article 

Google Scholar 
McCairns, R. J. S., Smith, S., Sasaki, M., Bernatchez, L. & Beheregaray, L. B. The adaptive potential of subtropical rainbowfish in the face of climate change: heritability and heritable plasticity for the expression of candidate genes. Evol. Appl. 9, 531–545 (2016).Article 
CAS 

Google Scholar 
McGuigan, K., Zhu, D., Allen, G. & Moritz, C. Phylogenetic relationships and historical biogeography of melanotaeniid fishes in Australia and New Guinea. Mar. Freshwat. Res. 51, 713–723 (2000).Article 

Google Scholar 
Unmack, P. J. et al. Malanda Gold: the tale of a unique rainbowfish from the Atherton Tablelands, now on the verge of extinction. Fish. Sahul. 30, 1039–1054 (2016).
Google Scholar 
Moritz, C. Strategies to protect biological diversity and the evolutionary processes that sustain it. Syst. Biol. 51, 238–254 (2002).Article 

Google Scholar 
Pope, L., Estoup, A. & Moritz, C. Phylogeography and population structure of an ecotonal marsupial, Bettongia tropica, determined using mtDNA and microsatellites. Mol. Ecol. 9, 2041–2053 (2000).Article 
CAS 

Google Scholar 
Hugall, A., Moritz, C., Moussalli, A. & Stanisic, J. Reconciling paleodistribution models and comparative phylogeography in the Wet Tropics rainforest land snail Gnarosophia bellendenkerensis (Brazier 1875). Proc. Natl Acad. Sci. USA 99, 6112–6117 (2002).Article 
CAS 

Google Scholar 
Moritz, C. et al. Identification and dynamics of a cryptic suture zone in tropical rainforest. Proc. R. Soc. B. 276, 1235–1244 (2009).Article 
CAS 

Google Scholar 
Phillips, B. L., Baird, S. J. & Moritz, C. When vicars meet: a narrow contact zone between morphologically cryptic phylogeographic lineages of the rainforest skink, Carlia rubrigularis. Evolution 58, 1536–1548 (2004).
Google Scholar 
Krosch, M. N., Baker, A. M., Mckie, B. G., Mather, P. B. & Cranston, P. S. Deeply divergent mitochondrial lineages reveal patterns of local endemism in chironomids of the Australian Wet Tropics. Austral Ecol. 34, 317–328 (2009).Article 

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

Google Scholar 
Whitehead, P. et al. Temporal development of the Atherton Basalt Province, north Queensland. Aust. J. Earth Sci. 54, 691–709 (2007).Article 
CAS 

Google Scholar 
Moy, K. G., Unmack, P. J., Lintermans, M., Duncan, R. P. & Brown, C. Barriers to hybridisation and their conservation implications for a highly threatened Australian fish species. Ethology 125, 142–152 (2019).Article 

Google Scholar 
Alexander, D. H., Novembre, J. & Lange, K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 19, 1655–1664 (2009).Article 
CAS 

Google Scholar 
Buerkle, C. A. Maximum‐likelihood estimation of a hybrid index based on molecular markers. Mol. Ecol. Notes 5, 684–687 (2005).Article 
CAS 

Google Scholar 
Anderson, E. & Thompson, E. A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160, 1217–1229 (2002).Article 
CAS 

Google Scholar 
Dorion, S. & Landry, J. Activation of the mitogen-activated protein kinase pathways by heat shock. Cell Stress Chaperones 7, 200 (2002).Article 
CAS 

Google Scholar 
Blumstein, M. et al. Protocol for projecting allele frequency change under future climate change at adaptive-associated loci. STAR Protoc. 1, 100061 (2020).Article 

Google Scholar 
Gougherty, A. V., Keller, S. R. & Fitzpatrick, M. C. Maladaptation, migration and extirpation fuel climate change risk in a forest tree species. Nat. Clim. Change 11, 166–171 (2021).Article 

Google Scholar 
Blumstein, M. et al. A new perspective on ecological prediction reveals limits to climate adaptation in a temperate tree species. Curr. Biol. 30, 1447–1453. e1444 (2020).Article 
CAS 

Google Scholar 
Razgour, O. et al. Considering adaptive genetic variation in climate change vulnerability assessment reduces species range loss projections. Proc. Natl Acad. Sci. USA 116, 10418–10423 (2019).Article 
CAS 

Google Scholar 
Goicoechea, P. G. et al. Adaptive introgression promotes fast adaptation in oaks marginal populations. Preprint available at bioRxiv https://doi.org/10.1101/731919 (2019).Lavergne, S. & Molofsky, J. Increased genetic variation and evolutionary potential drive the success of an invasive grass. Proc. Natl Acad. Sci. USA 104, 3883–3888 (2007).Article 
CAS 

Google Scholar 
De Carvalho, D. et al. Admixture facilitates adaptation from standing variation in the European aspen (Populus tremula L.), a widespread forest tree. Mol. Ecol. 19, 1638–1650 (2010).Article 

Google Scholar 
De-Kayne, R. et al. Genomic architecture of adaptive radiation and hybridization in Alpine whitefish. Nat. Commun. 13, 4479 (2022).Article 
CAS 

Google Scholar 
Baskett, M. L. & Gomulkiewicz, R. Introgressive hybridization as a mechanism for species rescue. Theor. Ecol. 4, 223–239 (2011).Article 

Google Scholar 
Meier, J. I. et al. The coincidence of ecological opportunity with hybridization explains rapid adaptive radiation in Lake Mweru cichlid fishes. Nat. Commun. 10, 1–11 (2019).Article 
CAS 

Google Scholar 
Svardal, H. et al. Ancestral hybridization facilitated species diversification in the Lake Malawi cichlid fish adaptive radiation. Mol. Biol. Evol. 37, 1100–1113 (2020).Article 
CAS 

Google Scholar 
Racimo, F., Sankararaman, S., Nielsen, R. & Huerta-Sánchez, E. Evidence for archaic adaptive introgression in humans. Nat. Rev. Genet. 16, 359–371 (2015).Article 
CAS 

Google Scholar 
Jeong, C. et al. Admixture facilitates genetic adaptations to high altitude in Tibet. Nat. Commun. 5, 1–7 (2014).Article 

Google Scholar 
Nolte, A. W., Freyhof, J., Stemshorn, K. C. & Tautz, D. An invasive lineage of sculpins, Cottus sp. (Pisces, Teleostei) in the Rhine with new habitat adaptations has originated from hybridization between old phylogeographic groups. Proc. R. Soc. B. 272, 2379–2387 (2005).Article 

Google Scholar 
Fitzpatrick, M. C., Chhatre, V. E., Soolanayakanahally, R. Y. & Keller, S. R. Experimental support for genomic prediction of climate maladaptation using the machine learning approach Gradient Forests. Mol. Ecol. Resour. 21, 2749–2765 (2021).Article 
CAS 

Google Scholar 
Schneider, C., Cunningham, M. & Moritz, C. Comparative phylogeography and the history of endemic vertebrates in the Wet Tropics rainforests of Australia. Mol. Ecol. 7, 487–498 (1998).Article 

Google Scholar 
Hewitt, G. M. Quaternary phylogeography: the roots of hybrid zones. Genetica 139, 617–638 (2011).Article 

Google Scholar 
Pfennig, K. S., Kelly, A. L. & Pierce, A. A. Hybridization as a facilitator of species range expansion. Proc. R. Soc. B. 283, 20161329 (2016).Article 

Google Scholar 
Soulé, M. E. What is conservation biology? A new synthetic discipline addresses the dynamics and problems of perturbed species, communities, and ecosystems. Bioscience 35, 727–734 (1985).
Google Scholar 
Biermann, C. & Havlick, D. Genetics and the question of purity in cutthroat trout restoration. Restor. Ecol. 29, e13516 (2021).Article 

Google Scholar 
Fredrickson, R. J. & Hedrick, P. W. Dynamics of hybridization and introgression in red wolves and coyotes. Conserv. Biol. 20, 1272–1283 (2006).Article 

Google Scholar 
Hirashiki, C., Kareiva, P. & Marvier, M. Concern over hybridization risks should not preclude conservation interventions. Conserv. Sci. Pract. 3, e424 (2021).
Google Scholar 
Unmack, P. J., Allen, G. R. & Johnson, J. B. Phylogeny and biogeography of rainbowfishes (Melanotaeniidae) from Australia and New Guinea. Mol. Phylogenet. Evol. 67, 15–27 (2013).Article 

Google Scholar 
Allen, G. Rainbowfishes in Nature and the Aquarium (Tetra Publications, 1995).Seehausen, O. Hybridization and adaptive radiation. Trends Ecol. Evol. 19, 198–207 (2004).Article 

Google Scholar 
Pusey, B., Kennard, M. J. & Arthington, A. H. Freshwater Fishes of North-eastern Australia (CSIRO Publishing, 2004).Zhu, D., Degnan, S. & Moritz, C. Evolutionary distinctiveness and status of the endangered Lake Eacham rainbowfish (Melanotaenia eachamensis). Conserv. Biol. 12, 80–93 (1998).Article 

Google Scholar 
McGuigan, K., Chenoweth, S. F. & Blows, M. W. Phenotypic divergence along lines of genetic variance. Am. Nat. 165, 32–43 (2005).Article 

Google Scholar 
Sunnucks, P. & Hales, D. F. Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae). Mol. Biol. Evol. 13, 510–524 (1996).Article 
CAS 

Google Scholar 
Peterson, B., Weber, J., Kay, E., Fisher, H. & Hoekstra, H. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PLoS ONE 7, e37135 (2012).Article 
CAS 

Google Scholar 
Catchen, J. M., Amores, A., Hohenlohe, P., Cresko, W. & Postlethwait, J. H. Stacks: building and genotyping loci de novo from short-read sequences. G3: Genes Genomes Genet. 1, 171–182 (2011).Article 
CAS 

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 
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357 (2012).Article 
CAS 

Google Scholar 
DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).Article 
CAS 

Google Scholar 
Danecek, P. et al. Twelve years of SAMtools and BCFtools. Gigascience 10, giab008 (2021).Article 

Google Scholar 
Goudet, J. Hierfstat, a package for R to compute and test hierarchical F‐statistics. Mol. Ecol. Notes 5, 184–186 (2005).Article 

Google Scholar 
R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2021).Bailey, R. ribailey/gghybrid: gghybrid R package for Bayesian hybrid index and genomic cline estimation. v2.0.0 https://doi.org/10.5281/zenodo.3676498 (2020).Wringe, B. hybriddetective: automates the process of detecting hybrids from genetic data. R package version 0.1.0.9000 https://github.com/bwringe/hybriddetective (2016).Pickrell, J. K. & Pritchard, J. K. Inference of population splits and mixtures from genome-wide allele frequency data. PLoS Genet. 8, e1002967 (2012).Article 
CAS 

Google Scholar 
Malinsky, M., Matschiner, M. & Svardal, H. Dsuite‐Fast D‐statistics and related admixture evidence from VCF files. Mol. Ecol. Resour. 21, 584–595 (2021).Article 

Google Scholar 
Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007).Article 
CAS 

Google Scholar 
Green, R. E. et al. A draft sequence of the Neandertal genome. Science 328, 710–722 (2010).Article 
CAS 

Google Scholar 
Durand, E. Y., Patterson, N., Reich, D. & Slatkin, M. Testing for ancient admixture between closely related populations. Mol. Biol. Evol. 28, 2239–2252 (2011).Article 
CAS 

Google Scholar 
Malinsky, M. et al. Genomic islands of speciation separate cichlid ecomorphs in an East African crater lake. Science 350, 1493–1498 (2015).Article 
CAS 

Google Scholar 
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).Article 
CAS 

Google Scholar 
Karger, D. N. et al. Climatologies at high resolution for the earth’s land surface areas. Sci. Data 4, 1–20 (2017).Article 

Google Scholar 
Karger, D. N. et al. CHELSA climatologies at high resolution for the Earth’s land surface areas (v.1.0). https://doi.org/10.1594/WDCC/CHELSA_v1 (2016).Ackerley, D. & Dommenget, D. Atmosphere-only GCM (ACCESS1.0) simulations with prescribed land surface temperatures. Geosci. Model Dev. 9, 2077–2098 (2016).Article 

Google Scholar 
Brown, J. L., Hill, D. J., Dolan, A. M., Carnaval, A. C. & Haywood, A. M. PaleoClim: high spatial resolution paleoclimate surfaces for global land areas. Sci. Data 5, 1–9 (2018).Article 

Google Scholar 
Fordham, D. A. et al. PaleoView: a tool for generating continuous climate projections spanning the last 21,000 years at regional and global scales. Ecography 40, 1348–1358 (2017).Article 

Google Scholar 
Thuiller, W., Lafourcade, B., Engler, R. & Araújo, M. B. BIOMOD–a platform for ensemble forecasting of species distributions. Ecography 32, 369–373 (2009).Article 

Google Scholar 
Lemus-Canovas, M., Lopez-Bustins, J. A., Martin-Vide, J. & Royé, D. synoptReg: an R package for computing a synoptic climate classification and a spatial regionalization of environmental data. Environ. Model. Softw. 118, 114–119 (2019).Article 

Google Scholar 
Hao, T., Elith, J., Guillera‐Arroita, G. & Lahoz‐Monfort, J. J. A review of evidence about use and performance of species distribution modelling ensembles like BIOMOD. Divers. Distrib. 25, 839–852 (2019).Article 

Google Scholar 
Galpern, P., Peres‐Neto, P. R., Polfus, J. & Manseau, M. MEMGENE: spatial pattern detection in genetic distance data. Methods Ecol. Evol. 5, 1116–1120 (2014).Article 

Google Scholar 
Peres‐Neto, P. R. & Galpern, P. memgene: spatial pattern detection in genetic distance data using Moran’s eigenvector maps. R package version 1.0.1 https://cran.r-project.org/web/packages/memgene/ (2019).Oksanen, J. et al. vegan: community ecology package. R package version 2.3–0 https://cran.r-project.org/web/packages/vegan/ (2015).Forester, B. R., Jones, M. R., Joost, S., Landguth, E. L. & Lasky, J. R. Detecting spatial genetic signatures of local adaptation in heterogeneous landscapes. Mol. Ecol. 25, 104–120 (2015).Article 

Google Scholar 
Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6, 80–92 (2012).Article 
CAS 

Google Scholar 
Szklarczyk, D. et al. The STRING database in 2021: customizable protein–protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res. 49, D605–D612 (2021).Article 
CAS 

Google Scholar 
Brauer, C. J. et al. Data for ‘Natural hybridisation reduces vulnerability to climate change’. figshare https://doi.org/10.6084/m9.figshare.21692918 (2022).Brauer, C. J. et al. Code for ‘Natural hybridisation reduces vulnerability to climate change’. GitHub https://github.com/pygmyperch/NER (2022). More

Vegetation and land-cover dataTo monitor vegetation at the global scale, we use three datasets: (1) vegetation optical depth (VOD, 0.25°, Ku-Band, daily 1987–201723) (Fig. 1A), (2) AVHRR GIMMSv3g normalized difference vegetation index (NDVI, 1/12°, bi-weekly 1981–201524) (Fig. 1B), and (3) MODIS MOD13 NDVI at 0.05° (16-day, 2000–202125). We correct for spurious values in the NDVI data (e.g., cloud contamination) using the method of Chen et al.43. We resample the VOD data using bi-weekly medians to agree with the NDVI data time sampling.For all three vegetation datasets, we remove seasonality and long-term trends using seasonal trend decomposition by Loess4,44 based on the proposed optimal parameters listed in Cleveland et al.44 (code available on Zenodo45). That is, we use a period of 24 (bi-monthly, 1 year), 47 for the trend smoother (just under 2 years) and 25 for low-pass (just over 1 year). We only use the STL residual—the de-seasoned and de-trended NDVI and VOD time series—in our analysis.To contextualize our understanding of vegetation resilience, we use MODIS MCD12Q1 land cover46 (Fig. 1C) as well as a global average aridity index based on WorldCLIM data31 (Fig. 1D). We exclude from our analysis anthropogenic and non-vegetated landscapes (e.g., permanent snow and ice, desert, urban), as well as any land covers which have changed (e.g., forest to grassland) during the period 2001–2020.Precipitation data and variability metricsTo measure precipitation at the global scale, we rely upon ERA5 data (~30 km, monthly, 1981–2021)33. We process global-scale precipitation metrics using the Google Earth Engine47 platform. We further use the sum of soil moisture from the surface down to 28 cm of depth (first two layers of the ECMWF Integrated Forecasting System soil moisture estimates) to quantify soil moisture means and inter-annual variability33.It is well-documented that vegetation resilience is responsive to the MAP of certain regions1. However, the role of precipitation variability in controlling vegetation resilience has not been well-studied. Here we examine precipitation variability in terms of both intra- and inter-annual patterns. Intra-annual precipitation variability is determined in terms of the Walsh-Lawler Seasonality index32 (Fig. 1D), calculated using monthly data from ERA533.Partly due to the fact that precipitation is non-negative, simple inter-annual variability metrics such as the standard deviation of annual precipitation sums are biased by the absolute precipitation sums; higher precipitation regions have a higher possible range of variability. To limit the influence of MAP, we hence investigate the standard deviation of annual precipitation sums normalized by the MAP, over the period 1981–2021, based on ERA5 data33 (Fig. 1F). We motivate our normalization by MAP with the strong linear relationship between MAP and MAP standard deviation (Supplementary Fig. S2). We further confirm our discovered relationships (Fig. 5) using only those regions where MAP was between the 40 and 60th percentile of MAP for a given land cover (Supplementary Figs. S11,S12). This serves as an additional check that our normalization of MAP standard deviation by MAP does not bias the inferred relationship between vegetation resilience and precipitation variability. Similarly, we generate a normalized inter-annual soil moisture variability by normalizing year-on-year soil moisture standard deviation (Supplementary Fig. S8) by long-term mean soil moisture (Supplementary Fig. S5).Empirical resilience estimationResilience is defined as the ability of a system to recover from perturbations, and can be quantified empirically by the speed of recovery to the previous state16,17. To measure resilience on the global scale, we employ a recently introduced methodology4 which we will briefly summarize in the following.We first identify sharp transitions in the vegetation time series using an 18-point (9 month) moving window to define local slopes throughout the time series48. We then identify slopes above the 99th percentile, and define connected regions as individual perturbations. The highest peak (largest instantaneous slope) within each connected region is then labeled as an individual disturbance.The employed approach does not delineate every rapid transition in a time series due to our reliance on percentiles; our dataset will be inherently biased towards the largest transitions. Furthermore, the same transitions are not guaranteed to be captured for both NDVI and VOD data in each location, as the percentiles will naturally vary between the datasets. Finally, our method will in some cases produce false positives, especially in cases where a given time series does not have any significant rapid transitions. To limit the influence of false positives on our results, we discard any perturbations where the time series does not drop significantly, and where the period before and after a given transition does not pass a two-sample Kolmogorov–Smirnov test4.Finally, using our global set of time-series transitions, we can identify each local vegetation (NDVI or VOD) minima, and use the five following years of data to fit an exponential function to the residual time series, assuming that the recovery after a perturbation to a vegetation state x0 follows approximately the equation$$x(t),approx ,{x}_{0}{e}^{rt}$$
(1)
where x(t) denotes the vegetation state at time t after the perturbation. Negative r indicates that the vegetation system will return to the original stable state at rate ∣r∣. For positive r, the initial perturbation would be amplified, suggesting a non-resilient vegetation state. Our empirical recovery rates are defined as the fitted exponent r, obtained for each detected transition in the NDVI and VOD residual time series. We finally use the coefficient of determination R2 to remove instances where the fitted exponential poorly matches the underlying data4.For the empirical estimate of the restoring rate obtained from fitting an exponential to the recovery after an abrupt negative deviation of VOD or NDVI, abrupt changes in the mean state induced by changing sensors rather than an actual vegetation shift may impact the results. However, all datasets used here are tightly cross-calibrated to eliminate mean-shifts when new instruments are introduced23,24. It is therefore unlikely that changes in the instrumentation of the various datasets unduly influence our empirical estimates of λ.Dynamical system metrics of resilienceThe lag-one autocorrelation (AC1) has previously been proposed to measure the stability of real-world dynamical systems in general, and the resilience of vegetation systems in particular1,19,20,21,49. Based on the concept of critical slowing down, the AC1 has, together with the variance, also been suggested as an early-warning indicator for forthcoming critical transitions50,51. Mathematically, the suitability of the variance and AC1 as resilience measures and early-warning indicators can be motivated as follows4,52,53. First, linearize the system around a given stable state x*:$$dbar{x}=lambda bar{x}dt+sigma dW$$
(2)
for (bar{x}: !!=x-{x}^{*}), assuming a Wiener Process W with standard deviation σ. The dynamics are stable for λ  More

Hoffmann, A. A. & Sgrò, C. M. Nature 470, 479–485 (2011).Article 
CAS 

Google Scholar 
Taylor, S. A. & Larson, E. L. Nat. Ecol. Evol. 3, 170–177 (2019).Article 

Google Scholar 
Brauer, C. J. et al. Nat. Clim. Change https://doi.org/10.1038/s41558-022-01585-1 (2023).Article 

Google Scholar 
Grinnell, J. Auk 34, 427–433 (1917).Article 

Google Scholar 
Peterson, A. T. et al. Ecological Niches and Geographic Distributions (Princeton Univ. Press, 2011).Wiens, J. A., Stralberg, D., Jongsomjit, D., Howell, C. A. & Snyder, M. A. Proc. Natl Acad. Sci. USA 106, 19729–19736 (2009).Article 
CAS 

Google Scholar 
Aguirre-Liguori, J. A., Ramírez-Barahona, S. & Gaut, B. S. Nat. Ecol. Evol. 5, 1350–1360 (2021).Article 

Google Scholar 
Fitzpatrick, M. C. & Keller, S. R. Ecol. Lett. 18, 1–16 (2015).Article 

Google Scholar 
Bay, R. A. et al. Science 359, 83–86 (2018).Article 
CAS 

Google Scholar 
Capblancq, T., Fitzpatrick, M. C., Bay, R. A., Exposito-Alonso, M. & Keller, S. R. Annu. Rev. Ecol. Evol. Syst. 51, 245–269 (2020).Article 

Google Scholar 
Rellstab, C., Dauphin, B. & Exposito‐Alonso, M. Evol. Appl. 14, 1202–1212 (2021).Article 

Google Scholar 
Allendorf, F. W., Leary, R. F., Spruell, P. & Wenburg, J. K. Trends Ecol. Evol. 16, 613–622 (2001).Article 

Google Scholar 
Rhymer, J. M. & Simberloff, D. Annu. Rev. Ecol. Syst. 27, 83–109 (1996).Article 

Google Scholar 
Todesco, M. et al. Evol. Appl. 9, 892–908 (2016).Article 
CAS 

Google Scholar  More

Davis, K. F., Downs, S. & Gephart, J. A. Towards food supply chain resilience to environmental shocks. Nat. Food 2, 54–65 (2021).Article 

Google Scholar 
Lempert, R. J. & Collins, M. T. Managing the risk of uncertain threshold responses: comparison of robust, optimum, and precautionary approaches. Risk Anal. 27, 1009–1026 (2007).Article 

Google Scholar 
Garnett, P., Doherty, B. & Heron, T. Vulnerability of the United Kingdom’s food supply chains exposed by COVID-19. Nat. Food 1, 315–318 (2020).Article 
CAS 

Google Scholar 
Abson, D. J. et al. Leverage points for sustainability transformation. Ambio 46, 30–39 (2017).Article 

Google Scholar 
Westley, F. et al. Tipping toward sustainability: emerging pathways of transformation. Ambio 40, 762–780 (2011).Article 

Google Scholar 
Steffen, W., Broadgate, W., Deutsch, L., Gaffney, O. & Ludwig, C. The trajectory of the Anthropocene: the Great Acceleration. Anthr. Rev. 2, 81–98 (2015).
Google Scholar 
Jouffray, J.-B., Blasiak, R., Norström, A. V., Österblom, H. & Nyström, M. The blue acceleration: the trajectory of human expansion into the ocean. One Earth 2, 43–54 (2020).Article 

Google Scholar 
Adger, W. N., Eakin, H. & Winkels, A. Nested and teleconnected vulnerabilities to environmental change. Front. Ecol. Environ. 7, 150–157 (2009).Article 

Google Scholar 
Nyström, M. et al. Anatomy and resilience of the global production ecosystem. Nature 575, 98–108 (2019).Article 

Google Scholar 
Mason, W. & Watts, D. J. Collaborative learning in networks. Proc. Natl Acad. Sci. USA 109, 764–769 (2012).Article 
CAS 

Google Scholar 
Helbing, D. Globally networked risks and how to respond. Nature 497, 51–59 (2013).Article 
CAS 

Google Scholar 
Worm, B. & Paine, R. T. Humans as a hyperkeystone species. Trends Ecol. Evol. 31, 600–607 (2016).Article 

Google Scholar 
Crutzen, P. J. & Stoermer, E. F. in The Future of Nature (eds Robin, L. et al.) 479–490 (Yale Univ. Press, 2017); https://doi.org/10.12987/9780300188479-041Ellis, E. C. Anthropogenic transformation of the terrestrial biosphere. Phil. Trans. R. Soc. A 369, 1010–1035 (2011).Article 

Google Scholar 
Senevirante, S. I. et al. in Climate Change 2021: The Physical Science Basis (eds Masson-Delmotte, V. et al.) 1513–1766 (IPCC, Cambridge Univ. Press, 2021).Frank, A. B. et al. Dealing with femtorisks in international relations. Proc. Natl Acad. Sci. USA 111, 17356–17362 (2014).Article 
CAS 

Google Scholar 
Folke, C. et al. Our future in the Anthropocene biosphere. Ambio 50, 834–869 (2021).Article 

Google Scholar 
Walker, B. & Salt, D. Resilience Practice: Building Capacity to Absorb Disturbance and Maintain Function (Island Press/Center for Resource Economics, 2012); https://doi.org/10.5822/978-1-61091-231-0Biggs, R., Schlüter, M. & Schoon, M. L. (eds) Principles for Building Resilience: Sustaining Ecosystem Services in Social–Ecological Systems (Cambridge Univ. Press, 2015); https://doi.org/10.1017/CBO9781316014240Cervantes Saavedra, M. de & Rutherford, J. Don Quixote: The Ingenious Hidalgo de la Mancha (Penguin, 2003).Coronese, M., Lamperti, F., Keller, K., Chiaromonte, F. & Roventini, A. Evidence for sharp increase in the economic damages of extreme natural disasters. Proc. Natl Acad. Sci. USA 116, 21450–21455 (2019).Article 
CAS 

Google Scholar 
Cottrell, R. S. et al. Food production shocks across land and sea. Nat. Sustain. 2, 130–137 (2019).Article 

Google Scholar 
Elmqvist, T. et al. Response diversity, ecosystem change, and resilience. Front. Ecol. Environ. 1, 488–494 (2003).Article 

Google Scholar 
Arrow, K. J. & Fisher, A. C. Environmental preservation, uncertainty, and irreversibility. Q. J. Econ. 88, 312–319 (1974).Article 

Google Scholar 
Dixit, A. K. & Pindyck, R. S. Investment under Uncertainty (Princeton Univ. Press, 1994).Markowitz, H. Portfolio selection. J. Finance 7, 77–91 (1952).
Google Scholar 
Sharpe, W. F. Capital asset prices: a theory of market equilibrium under conditions of risk. J. Finance 19, 425–442 (1964).
Google Scholar 
Cifdaloz, O., Regmi, A., Anderies, J. M. & Rodriguez, A. A. Robustness, vulnerability, and adaptive capacity in small-scale social–ecological systems: the Pumpa Irrigation System in Nepal. Ecol. Soc. 15, art39 (2010).Article 

Google Scholar 
Levin, S. A. et al. Governance in the face of extreme events: lessons from evolutionary processes for structuring interventions, and the need to go beyond. Ecosystems 25, 697–711 (2022).Article 

Google Scholar 
Peterson, G., Allen, C. R. & Holling, C. S. Ecological resilience, biodiversity, and scale. Ecosystems 1, 6–18 (1998).Article 

Google Scholar 
Nyström, M. Redundancy and response diversity of functional groups: implications for the resilience of coral reefs. Ambio 35, 30–35 (2006).Article 

Google Scholar 
Kummu, M. et al. Interplay of trade and food system resilience: gains on supply diversity over time at the cost of trade independency. Glob. Food Secur. 24, 100360 (2020).Article 

Google Scholar 
Hedblom, M., Andersson, E. & Borgström, S. Flexible land-use and undefined governance: from threats to potentials in peri-urban landscape planning. Land Use Policy 63, 523–527 (2017).Article 

Google Scholar 
Haldane, A. Rethinking the Financial Network—Speech by Andy Haldane (Bank of England, 2009); https://www.bankofengland.co.uk/speech/2009/rethinking-the-financial-networkHaldane, A. G. & May, R. M. Systemic risk in banking ecosystems. Nature 469, 351–355 (2011).Article 
CAS 

Google Scholar 
Carpenter, S. R., Brock, W. A., Folke, C., van Nes, E. H. & Scheffer, M. Allowing variance may enlarge the safe operating space for exploited ecosystems. Proc. Natl Acad. Sci. USA 112, 14384–14389 (2015).Article 
CAS 

Google Scholar 
Mouillot, D., Graham, N. A. J., Villéger, S., Mason, N. W. H. & Bellwood, D. R. A functional approach reveals community responses to disturbances. Trends Ecol. Evol. 28, 167–177 (2013).Article 

Google Scholar 
Leslie, P. & McCabe, J. T. Response diversity and resilience in social–ecological systems. Curr. Anthropol. 54, 114–143 (2013).Article 

Google Scholar 
Biggs, R. et al. Toward principles for enhancing the resilience of ecosystem services. Annu. Rev. Environ. Resour. 37, 421–448 (2012).Article 

Google Scholar 
Anderies, J. M. Managing variance: key policy challenges for the Anthropocene. Proc. Natl Acad. Sci. USA 112, 14402–14403 (2015).Article 
CAS 

Google Scholar 
Csete, M. E. & Doyle, J. C. Reverse engineering of biological complexity. Science 295, 1664–1669 (2002).Article 
CAS 

Google Scholar 
Carlson, J. M. & Doyle, J. Highly optimized tolerance: robustness and design in complex systems. Phys. Rev. Lett. 84, 2529–2532 (2000).Article 
CAS 

Google Scholar 
Kitano, H. Biological robustness. Nat. Rev. Genet. 5, 826–837 (2004).Article 
CAS 

Google Scholar 
Csete, M. & Doyle, J. Bow ties, metabolism and disease. Trends Biotechnol. 22, 446–450 (2004).Article 
CAS 

Google Scholar 
Anderies, J. M., Rodriguez, A. A., Janssen, M. A. & Cifdaloz, O. Panaceas, uncertainty, and the robust control framework in sustainability science. Proc. Natl Acad. Sci. USA 104, 15194–15199 (2007).Article 
CAS 

Google Scholar 
Rodriguez, A. A., Cifdaloz, O., Anderies, J. M., Janssen, M. A. & Dickeson, J. Confronting management challenges in highly uncertain natural resource systems: a robustness–vulnerability trade-off approach. Environ. Model. Assess. 16, 15–36 (2011).Article 

Google Scholar 
Charpentier, A. Insurability of climate risks. Geneva Pap. Risk Insur. Issues Pract. 33, 91–109 (2008).Article 

Google Scholar 
Alfieri, L., Feyen, L. & Di Baldassarre, G. Increasing flood risk under climate change: a pan-European assessment of the benefits of four adaptation strategies. Climatic Change 136, 507–521 (2016).Article 

Google Scholar 
Isakson, S. R. Derivatives for development? Small-farmer vulnerability and the financialization of climate risk management: small-farmer vulnerability and financialization. J. Agrar. Change 15, 569–580 (2015).Article 

Google Scholar 
Müller, B. & Kreuer, D. Ecologists should care about insurance, too. Trends Ecol. Evol. 31, 1–2 (2016).Article 

Google Scholar 
Walker, B. et al. Looming global-scale failures and missing institutions. Science 325, 1345–1346 (2009).Article 
CAS 

Google Scholar 
Berkes, F. et al. Globalization, roving bandits, and marine resources. Science 311, 1557–1558 (2006).Article 
CAS 

Google Scholar 
Walker, B. H., Langridge, J. L. & McFarlane, F. Resilience of an Australian savanna grassland to selective and non-selective perturbations. Austral Ecol. 22, 125–135 (1997).Article 

Google Scholar 
Polasky, S. et al. Corridors of clarity: four principles to overcome uncertainty paralysis in the Anthropocene. BioScience 70, 1139–1144 (2020).Article 

Google Scholar 
Engström, G. et al. Carbon pricing and planetary boundaries. Nat. Commun. 11, 4688 (2020).Article 

Google Scholar 
Sun, J. C., Ugolini, S. & Vivier, E. Immunological memory within the innate immune system. EMBO J. https://doi.org/10.1002/embj.201387651 (2014).Vély, F. et al. Evidence of innate lymphoid cell redundancy in humans. Nat. Immunol. 17, 1291–1299 (2016).Article 

Google Scholar 
Grimm, N., Cook, E., Hale, R. & Iwaniec, D. in The Routledge Handbook of Urbanization and Global Environmental Change (eds Seto, K. et al.) Ch. 14 (Routledge, 2015).Jiang, B., Mak, C. N. S., Zhong, H., Larsen, L. & Webster, C. J. From broken windows to perceived routine activities: examining impacts of environmental interventions on perceived safety of urban alleys. Front. Psychol. 9, 2450 (2018).Article 

Google Scholar 
Andersson, E. et al. Urban climate resilience through hybrid infrastructure. Curr. Opin. Environ. Sustain. 55, 101158 (2022).Article 

Google Scholar 
Douglas, M. & Wildavsky, A. Risk and Culture: An Essay on the Selection of Technological and Environmental Dangers (Univ. of California Press, 1983).Weber, E. U., Ames, D. R. & Blais, A.-R. ‘How do I choose thee? Let me count the ways’: a textual analysis of similarities and differences in modes of decision-making in China and the United States. Manage. Organ. Rev. 1, 87–118 (2005).Article 

Google Scholar 
Kunreuther, H. et al. in Climate Change 2014: Mitigation of Climate Change (eds Edenhofer, O. et al.) Ch. 2 (IPCC, Cambridge Univ. Press, 2014); https://www.ipcc.ch/site/assets/uploads/2018/02/ipcc_wg3_ar5_chapter2.pdfMeadows, D. H. Thinking in Systems: A Primer (Earthscan, 2009).Nyborg, K. et al. Social norms as solutions. Science 354, 42–43 (2016).Article 
CAS 

Google Scholar 
Hall, P. A. & Lamont, M. (eds) Social Resilience in the Neoliberal Era (Cambridge Univ. Press, 2013).Norström, A. V. et al. Principles for knowledge co-production in sustainability research. Nat. Sustain. 3, 182–190 (2020).Article 

Google Scholar 
United Nations Conference on Trade and Development Review of Maritime Transport 2017 (United Nations, 2017).United Nations Conference on Trade and Development Review of Maritime Transport 2018 (United Nations, 2019).Bailey, R. & Wellesley, L. Chatham House Report 2017: Chokepoints and Vulnerabilities in Global Food Trade (Energy, Environment and Resources Department, Chatham House, The Royal Institute of International Affairs, 2017); https://www.chathamhouse.org/sites/default/files/publications/research/2017-06-27-chokepoints-vulnerabilities-global-food-trade-bailey-wellesley-final.pdfKhoury, C. K. et al. Increasing homogeneity in global food supplies and the implications for food security. Proc. Natl Acad. Sci. USA 111, 4001–4006 (2014).Article 
CAS 

Google Scholar 
Hendrickson, M. K. Resilience in a concentrated and consolidated food system. J. Environ. Stud. Sci. 5, 418–431 (2015).Article 

Google Scholar 
Öborn, I. et al. Restoring rangelands for nutrition and health for humans and livestock. in The XXIV International Grassland Congress / XI International Rangeland Congress (Sustainable Use of Grassland and Rangeland Resources for Improved Livelihoods) (ed. National Organizing Committee of 2021 IGC/IRC Congress) (Kenya Agricultural and Livestock Research Organization, 2022).Vulnerable Supply Chains—Interim Report (Productivity Commission, Australian Government, 2021); https://www.pc.gov.au/inquiries/completed/supply-chains/interim More

Habitat loss is one of main threats to biodiversity worldwide and in general is perceived as something to be avoided. However, the prevalence of negative effects of forest fragmentation is less clear. Fragmentation creates edges between once-pristine forest and the adjacent non-forest system or systems (for example, agricultural lands, cities or water reservoirs), but the effects of these edges on biodiversity are not always clear. By performing a robust study of the interaction between insect pollinators and flowering plants at forest edges and within the forest, Ren et al.1 add a new piece to this puzzle by showing that forest edges can have a positive buffering effect on interaction networks. More

A small number of C. rodentium founders initiates enteric infectionTo enable monitoring of the pathogen population’s diversity during infection, we introduced short, random, ~20 nucleotide DNA tags (barcodes) at a neutral location in the C. rodentium genome. As previously described5, monitoring barcode diversity using high-throughput DNA sequencing and the STAMP (Sequence Tag-based Analysis of Microbial Populations) computational framework can quantify the constriction of the pathogen population that often occurs during establishment of infection (schematized in Fig. 1a). We created two independent STAMP libraries of barcoded bacteria. Library “STAMP-CR253” contains 253 unique barcodes integrated in the intergenic region between genes ROD_05521 and selU. The neutrality of the barcode insertions was confirmed by measuring growth in lysogeny broth (LB; Supplemental Fig. 1a). Library “STAMP-CR69K” contains approximately 69,000 unique barcodes inserted into the genome on a Tn7 vector, which integrates at a neutral site downstream of the glmS gene6,7. While the libraries were not directly compared, both yielded similar results in our studies.To validate that these barcoded STAMP libraries can quantify the population effects of a bottleneck, we created in vitro bottlenecks by plating serial dilutions of the libraries grown in culture. The number of colony forming units (CFU) per plate provides a true measure of the number of founders, i.e., the number of cells from the initial population (culture) that gave rise to the observed population (plated colonies). Bacteria were harvested from the plates, the barcodes were amplified and sequenced, and barcode frequencies were analyzed using the recently updated STAMP analysis pipeline “STAMPR”8. The size of the founding population (founders) was calculated by comparing the diversity and frequency of barcodes recovered from plated samples to those in the initial cultures. There was a strong correlation between the counted founders (CFU) and the calculated founders (Nr for STAMP-CR253 and Ns for STAMP-CR69K) up to 104 founders (Supplemental Fig. 1b). These data were also used as standard curves to increase the resolution of the experiments described below to approximately 106 founders.Contraction of a barcoded population during colonization changes the frequency and number of barcodes relative to the inoculum. To determine when a C. rodentium infection is founded, C57BL/6 J (B6) mice were orally gavaged with 4 × 108 CFUs (enumerated by serial dilution and plating). Remarkably, despite this relatively large dose, within 24 hours (h) there was an average of only 9 founders (geometric mean), and as few as one founder per mouse (a single barcode; Fig. 1b). Thus, only ~1 of every 4 × 107 cells in the inoculum establishes infection, revealing that host bottlenecks result in a massive constriction of the pathogen population. Beyond 24 h, the founding population remained stable at ~10 founders. The diminutive C. rodentium founding population indicates that the vast majority of the inoculum does not survive to give rise to detectable offspring and is thus either killed by the host or passes through the intestine and is excreted in feces. Consistent with the latter possibility, 5 h after inoculation there were 1 × 107 CFU C. rodentium per gram of feces with 9×104 founders, suggesting that at this early point a numerous and diverse population has already reached the colon and cecum but failed to become founders. Surprisingly, the contraction of the pathogen population continued beyond 5 h, when the pathogen had already reached the principal sites of colonization. Despite the profound bottleneck to infection, the ~10 founders were capable of replication, and by 5 days post inoculation the C. rodentium burden in the feces was on average 9 × 108 CFU/gram. Together, these observations reveal that there is a severe bottleneck to infection with this natural, mouse enteric pathogen; however, even though the restrictive bottleneck leaves a founding population that is a miniscule fraction of the inoculum, the founders robustly replicate, creating a total pathogen burden that ultimately exceeds the inoculum (Fig. 1b).The size of the founding population increases with doseWe reasoned that determining how dose impacts the number of founders could provide insight into the mechanisms underlying the bottleneck9,10. For example, one explanation proposed for the C. rodentium bottleneck is that it is created by finite niches or resources (e.g., sugar or amino acids) whose scarcity limits the size of the population11,12. At doses where the pathogen saturates this limited resource, the ‘finite resource’ hypothesis predicts that increasing dose will not increase the number of founders (schematized in Fig. 2a). An alternate possibility is that the bottleneck eliminates potential founders through a mechanism such as acid killing in the stomach13,14, which is expected to result in a founding population that increases with dose.Fig. 2: The C. rodentium bottleneck is defined by a fractional relationship between dose and founders.a Models for the relationship between dose and founding population. In the absence of a bottleneck, all bacteria from the inoculum become founders. If the inoculum contracts due to the limited availability of finite nutrients or niches (e.g. iron, sugar, binding sites), the diversity of the population and thus the size of the founding population will remain fixed once those nutrients are saturated. If increasing dose increases the number of founders, then the underlying mechanism is not due to a limited resource; instead, the bottleneck acts proportionally on the inoculum by eliminating potential founders. b, c C57BL/6 J mice were inoculated with doses ranging from 107 to 1010 CFU of STAMP-CR253 and the C. rodentium population was monitored in the feces (geometric means and standard deviations; ND not detected counted as 0.5). Additional shedding analysis in Supplemental Fig. 2. c The bottleneck impeding B6 colonization is described 5 days post inoculation by comparing dose and founders with a linear regression of the log10-transformed data (regression line with 95% confidence intervals; not detected counted as 0.8; x-intercept “ID50” 107.2–107.9 CFU). 4–8 animals per dose. Source data are provided as a Source Data file.Full size imageTo characterize the bottleneck, we orally inoculated B6 mice with C. rodentium doses ranging 1000-fold from 107 to 1010 CFUs. Doses (ge)108 CFUs led to infection, with the founding population decreasing for ~2 days before reaching a steady value that persisted until the infection began clearing, as indicated by a simultaneous decrease in the total population (burden) and founding population (Fig. 2b). Lower doses of C. rodentium resulted in fewer founders and a longer period to reach peak shedding, with a correspondingly longer time from inoculation to pathogen elimination. As the delay in shedding at lower doses correlated with the delay in clearance, all mice were infected for a similar number of days and had similar total fecal burdens, regardless of dose (Supplemental Fig. 2).The founding population was small in number. Even at the maximum inoculum of 1010 CFUs relatively few founders were detected (83, geometric mean; Fig. 2b). While founders were never numerous, increasing the size of the inoculum always increased the size of the founding population. The bottleneck eliminated a proportion of the C. rodentium population, resulting in a founding population that scaled with dose (increasing dose 100-fold also increased founders ~100-fold). These observations indicate that the number of founders is likely not dictated by limited space or resources, contradicting the finite resource hypothesis (Fig. 2a).As an increase in dose resulted in a proportional increase in the number of founders, we represented their relationship as a line by plotting log10-transformed dose and founding population data from 5 days post inoculation (Fig. 2c). This line indicates that the bottleneck is not fixed, but rather functions by eliminating a fraction of potential founders, as schematized in Fig. 2a (‘elimination bottleneck’). Since our findings conform to a simple fractional relationship between dose and founding population, we will use this relationship to define the bottleneck: in B6 mice 1 of every ~108 inoculated C. rodentium establish a replicative niche.The x-intercept of the log-linear relationship between dose and founders can be used to calculate the dose at which we expect 1 founder. This dose corresponds to the ID50 – the dose that leads to infection of ~50% of animals. Thus, for C. rodentium infection, the ID50, a critical parameter describing a pathogen’s infectivity, is a property biologically defined by the infection bottleneck. For B6 mice, the x-intercept of this line is between 107.2 and 107.9 CFU (95% confidence-intervals) and explains why infection did not result from an inoculum of 107 CFU (Fig. 2b). Surprisingly, even though C. rodentium is a natural mouse pathogen, at least ~100-million organisms are required to routinely establish infection.Stomach acid contributes a 10- to 100-fold bottleneck to C. rodentium colonizationWe next probed the contribution of stomach acid to the highly restrictive B6 enteric colonization bottleneck. The acidity of the stomach is thought to be a potent barrier against ingested bacteria; human studies find that taking stomach acid reducing drugs increases the risk of contracting multiple enteric pathogens15. Notably, it has been observed that eliminating stomach acid decreases the minimum infectious dose for C. rodentium and increases the size of the founding population13,14. Further, acid is mechanistically consistent with the fractional relationship which we observe between dose and founding population (Fig. 2). To test the role of stomach acid in restricting C. rodentium enteric colonization, we treated mice with the fast-acting, irreversible H2-antagonist Loxtidine (aka Lavoltidine)16. 3–5 h after Loxtidine treatment, the pH of the stomach rose from 2.5 to 4.7 (Fig. 3a). Importantly, a pH of 2.5 sterilized 1010 CFUs of C. rodentium in under 15 minutes (min), whereas pH 4.7 did not kill C. rodentium even after a 1 h exposure (Fig. 3b).Fig. 3: Stomach acid constricts the C. rodentium population by 10- to 100-fold.a The effect of Loxtidine on stomach acid in C57BL/6 J mice 3–5 h after intraperitoneal administration of 1 mg in 0.1 ml PBS. pH determined post-mortem in aspirated stomach fluid. Boxes arithmetic mean (2.5 for mock and 4.7 for Loxtidine). Two-tailed t test with p-value 0.0002. Animals are 7 (vehicle) and 5 (Loxtidine). b The acid tolerance of STAMP-CR69K in culture measured by diluting cells in LB at pH 2.5 or 4.7 and incubating at 37 °C with shaking. pH 2.5 sterilized 1010 CFU in 15 min. c, d 3–5 h after intraperitoneal administration of PBS (vehicle) or Loxtidine (1 mg), C57BL/6 J mice were orally gavaged with doses ranging from 107 to 1010 CFU of STAMP-CR69K. c Bacterial burden monitored in the feces for 5 days following inoculation (geometric means and standard deviations; ND not detected counted as 0.5). d Bottleneck to colonization measured 5 days post inoculation by comparing dose and founders with linear regression of the log10-transformed data (regression line and 95% confidence intervals; significance compares elevation with p-value 5.5 × 10−7; not detected counted as 0.8). 4 animals per dose per group. Source data are provided as a Source Data file.Full size imageLoxtidine treatment prior to inoculating B6 mice resulted in infection at a lower dose, a higher pathogen burden in the feces 1 day post inoculation, and more founders on day 5 (Fig. 3c, d). The fractional relationship between dose and founding population was also observed in the absence of stomach acid, but the line depicting this relationship was shifted upward. Loxtidine treatment increased the number of C. rodentium founders approximately 10-fold at every dose, reducing the ID50 computed from the founding population from 107.3 to 105.4 CFUs. Thus, stomach acid significantly contributes to the bottleneck restricting C. rodentium colonization. However, the magnitude of stomach acid’s contribution is relatively small, between 10- and 100-fold of the observed ~108-fold B6 bottleneck to C. rodentium colonization. In the absence of stomach acid, the C. rodentium population constricts >106-fold prior to establishing a replicative niche, indicating that other factors must more potently contribute to the bottleneck.Constriction of the C. rodentium inoculum occurs distal to the stomach, at the sites of infectionTo further define the C. rodentium population dynamics and host barriers that accompany establishment of infection, we probed where and when the bottleneck occurs. Five days post-inoculation, the largest pathogen burdens were detected in the cecum and distal colon, with less numerous populations in the small intestine (SI) (Fig. 4a), consistent with previous observations17. Within individual mice (intra-mouse) the cecum, colon, and feces contained related populations of C. rodentium, with approximately the same number of founders and similar barcodes (Fig. 4b–e). Importantly, the near identity of the barcodes found in the fecal population to those in the cecum and colon indicates that fecal samples can be used to report on the pathogen population at these primary infection sites, facilitating longitudinal monitoring. While intra-mouse populations were related, comparisons of barcodes between cohoused mice (inter-mouse) inoculated with the same inoculum revealed that each mouse contained a distinct C. rodentium population (Fig. 4c–e). The distinct identities of the founding populations in each of five cohoused, co-inoculated mice was apparent when comparing pathogen barcode frequencies with principal component analysis (PCA), where intra-mouse samples formed their own tight clusters (Fig. 4c). Similarly, analysis of barcode genetic distances showed that the intra-mouse pathogen populations were highly similar (low genetic distance), whereas they were dissimilar to the populations in cohoused, co-inoculated mice (Fig. 4d, e). A notable exception were the C. rodentium populations from some SI samples that were more closely related to cage-mates than other intra-mouse samples, likely reflecting recent inter-mouse exchange via coprophagy (Supplemental Fig. 3). These data suggest that despite the consumption of C. rodentium-laden feces, C. rodentium infection leads to super-colonization resistance at the primary infection sites in the cecum and colon, preventing transmission to cohoused, co-infected mice.Fig. 4: Infection is initiated by related populations of C. rodentium in the cecum and colon.a–e C. rodentium populations in whole organ homogenates from 5 cohoused (intra-cage) C57BL/6 J mice 5 days post inoculation with 4 × 108 CFU of STAMP-CR253. Within a mouse (intra-mouse) the C. rodentium populations at the primary sites of colonization (cecum, proximal colon, distal colon, feces) share founders (number, identity, and frequency of barcodes). a, b Lines connect intra-mouse samples. c Clustering of barcode populations by principal component analysis (PCA). d, e Relatedness determined by comparing the barcode frequencies by genetic distance (arithmetic means) with zero indicating no difference between populations (identical). e Two-tailed t test with p-value 5.8 × 10−48. p proximal, m mid, d distal, SI small intestine. Heatmap depicting genetic distance relationships of all intra-cage populations in Supplemental Fig. 3. Source data are provided as a Source Data file. f, g To determine when/where C. rodentium establishes a replicative niche, C57BL/6 J mice were orally gavaged with between 3 × 109 and 6 × 109 CFU STAMP-CR253. Following dissection, the cecum and colon were flushed to separate organ adherent (f) and luminal (g) bacteria. Burden and founders display geometric means and standard deviations. Bacteria not detected (ND) counted as 0.5. Relatedness of populations was determined by comparing the barcode frequencies of colon and cecal populations from within the same animal (intra-mouse) by genetic distance (arithmetic mean and standard deviation). 22 animals. Source data are provided as a Source Data file. h Model depicting how related C. rodentium populations could initiate infection in both the cecum and colon: (1) the inoculum minorly constricts passing through the stomach and SI to deposit diverse populations in the cecum and colon, (2) the populations in the cecum and colon contract separately over the first 24–48 h becoming dissimilar, and then (3) expansion occurs in either the cecum or colon moving to both locations. We depict the movement from cecum to colon as we judge this to be more likely, but the opposite is possible.Full size imageTo test this super-colonization resistance hypothesis, we separately infected two groups of ‘seed’ mice with different sets (A and B) of barcoded C. rodentium (Supplemental Fig. 4a). At the peak of colonization in the seed mice, 7 days post-inoculation, they were cohoused for 16 h along with an uninfected ‘contact’ mouse, three mice per cage. After 16 h, the mice were separated back into 3 cages containing mice originally inoculated with the A barcodes, inoculated with the B barcodes, or uninoculated. No transmission of barcodes was detected between the animals originally inoculated with the A and B barcodes (Supplemental Fig. 4b), confirming that C. rodentium infection prevents super-colonization. In marked contrast, the contact mice became infected with founders from seed A and/or B, demonstrating the ready transmission of C. rodentium from infected to uninfected mice. Furthermore, the co-infection of contact mice with barcodes from A and B confirms a previous report from super-infection experiments in mice lacking a microbiota18 that immunity to super-colonization takes time, providing a window for co-colonization. Importantly, super-colonization resistance indicates that founders are more likely to originate from the inoculum than other cohoused, infected animals.Based on the high burdens of C. rodentium in the cecum but not the colon during the first 3 days following inoculation, prior studies proposed that infection begins with pathogen expansion in the cecum, followed by subsequent spread to the colon17; a hypothesis that is consistent with the closely related intra-mouse C. rodentium populations that we observe in the cecum and colon 5 days after inoculation (Fig. 4a–e). To determine when and where C. rodentium initiates infection, we monitored the luminal and adherent C. rodentium populations in the cecum and colon. Within the first 5 h a large burden ( >107 CFU) and numerous founders ( >105 Nr’) were detected in both locations (Fig. 4f, g). Since a large founding population was observed in the cecum and colon early after inoculation, we can discount the model that the primary bottleneck occurs proximal to these locations (e.g., stomach acid or bile). The number of founders and the total burden contracted over the first 24 h, resulting in small (0.4) populations in the cecum and colon one day post inoculation. Expansion was detected first in the cecum, on day 2. Concomitant with cecal expansion, the populations in the cecum and colon became increasingly similar; i.e., the genetic distance between the populations became smaller. The most plausible model to fit these data (depicted in Fig. 4h) is that (1) within hours many bacteria pass through the stomach, reaching the cecum and colon, and then (2) these populations diverge as they separately constrict, and finally, (3) spread to both locations when a small number of founders begin to replicate. We propose that the initial population expansion begins in the cecum and then spreads to the colon, but we cannot rule out the opposite directionality because we were unable to serially sample the internal populations from a single mouse. However, displacement of the cecal population by bacteria from the colon seems unlikely because it would require non-flagellated C. rodentium to move against the bulk flow of the gut and thus we favor the model that infection initiates in the cecum.C3H/HeOuJ mice have a less restrictive bottleneck than C57BL/6 JWe next interrogated the host’s contribution to the bottleneck impeding C. rodentium colonization by quantifying the bottleneck in a more disease susceptible genotype of mice. While C. rodentium causes self-limited diarrhea in B6 mice, infection leads to a lethal diarrheal disease in C3H/HeOuJ (C3Ou) mice (Fig. 5a, b)19. We found that increased vulnerability to disease correlated with a less restrictive bottleneck. C. rodentium is 10- to 100-fold more infectious in C3Ou than B6 mice, infecting at a ~10-fold lower dose and producing ~10-times more founders at every dose (Fig. 5c). While the bottleneck was relaxed in C3Ou mice, a fractional relationship remained between dose and founding population, suggesting a similar underlying mechanism restricts colonization in both mouse genotypes. Also, as in B6 animals, higher doses and more founders accelerated the dynamics of pathogen shedding in C3Ou mice (Fig. 5a). These observations demonstrate that in addition to dose, the size of the founding population is determined in part by host genetics, which may impact the bottleneck through several mechanisms. Notably, changing host genotype caused a more lethal disease while only alleviating ~10-fold of the ~108-fold B6 bottleneck.Fig. 5: Host genotype impacts the bottleneck to C. rodentium colonization.C3H/HeOuJ (a) or C57BL/6 J (b) mice were inoculated with doses ranging from 106 to 1010 CFU of STAMP-CR253. The C. rodentium population was monitored in the feces (geometric means and standard deviations; ND not detected counted as 0.5) and animal health assessed by weight loss (percent compared to pre-inoculation; arithmetic means and standard deviations) and body condition. For survival, lines are percent of initial animals not moribund. c The bottleneck to C3Ou and B6 colonization is described 5 days post inoculation by comparing dose and founders with a linear regression of the log10-transformed data (regression line with 95% confidence intervals; significance compares elevation with p-value 5.5 × 10−7; not detected counted as 0.8). B6 bottleneck data is repeated from Fig. 2. 4 animals per dose. Source data are provided as a Source Data file.Full size imageThe bottleneck to C. rodentium enteric colonization is microbiota dependentAs shown above, a large portion of the restrictive, fractional, B6 bottleneck to C. rodentium colonization occurs distal to the stomach, at the chief sites of infection in the cecum and colon. These data strongly suggest that the principal step limiting colonization occurs during the pathogen’s establishment of a replicative niche in the cecum and/or colon. One factor present at these sites and previously linked to limiting C. rodentium colonization is the microbiota12,18. We therefore tested whether acute microbiota depletion eliminated the bottleneck to C. rodentium colonization. Treating mice with the antibiotic streptomycin for the 3 days prior to inoculation with streptomycin-resistant C. rodentium greatly accelerated pathogen population expansion, with mice shedding >109 CFUs per gram of feces within the first day (Fig. 6a). Further, streptomycin pretreatment almost completely ablated the bottleneck, with colonization at doses as low as ~100 CFUs; at this low dose, we measured an average of 25 founders 5 days post inoculation, indicating that C. rodentium experiences less than a 10-fold bottleneck following microbiota depletion (Fig. 6b). Significantly, streptomycin treatment does not alter the acidity of the animal’s stomach (Supplemental Fig. 5). Since an ~10-fold bottleneck remains after microbiota depletion and an ~10-fold bottleneck is stomach acid dependent (Fig. 3d), these data suggest that the combination of the microbiota and stomach acid can account for the majority of factors restricting C. rodentium colonization.Fig. 6: Streptomycin treatment ablates most of the bottleneck preventing C. rodentium colonization.The microbiota of conventional C57BL/6 J mice was reduced by treatment with the antibiotic streptomycin for the 3 days prior to inoculation with a streptomycin resistant library of STAMP-CR69K. a Founding population and bacterial burden monitored in the feces, and (b, c) the bottleneck to colonization measured by comparing dose and founders (geometric means and standard deviations; resolution limit is ~106 founders). (a) Animal health monitored by weight loss (percent compared to pre-inoculation; arithmetic means and standard deviations) and body condition. For survival, lines are percent of initial animals not moribund. Untreated B6 bottleneck data is repeated from Fig. 2 for comparison. 4 animals per dose. Streptomycin treatment does not impact stomach acidity (Supplemental Fig. 5). Source data are provided as a Source Data file.Full size imageTo confirm that streptomycin’s ablation of the bottleneck to C. rodentium colonization occurs because of microbiota depletion rather than an off-target effect, we also determined the bottleneck in B6 mice lacking a microbiota (germ-free). In germ-free mice, like streptomycin pretreated animals, there was almost no bottleneck to C. rodentium colonization (Fig. 7a, b). Animals lacking a microbiota were colonized at a dose of 150 CFU and shed numerous C. rodentium within 1 day of inoculation. Together, experiments with germ-free and streptomycin-pretreated mice reveal that the primary barrier to enteric colonization is linked to the microbiota.Fig. 7: The bottleneck to C. rodentium colonization is microbiota dependent.Germ-free C57BL/6 J mice were orally inoculated with doses ranging from 102 to 1010 CFU of STAMP-CR69K. 4 animals per dose, cohoused with animals receiving the same dose in sterile cages. Measurement of germ-free stomach acidity in Supplemental Fig. 5. Source data are provided as a Source Data file. a Bacterial burden and founding population monitored in the feces (geometric means and standard deviations; resolution limit is ~106 founders). Animal health monitored by weight loss (percent compared to pre-inoculation; arithmetic means and standard deviations) and body condition. No animals became moribund. b, c Bottleneck to colonization measured by comparing dose and founders (geometric means and standard deviations). SPF B6 bottleneck data is repeated from Fig. 2 for comparison. d To determine if colonization was accompanied by changes in the C. rodentium genome, whole genome sequencing was performed on 3 clones (colonies) from the STAMP-CR69K input library and compared to clones isolated from feces of infected mice (1 colony per mouse). Boxes represent the genome status of the LEE pathogenicity island. No LEE genomic changes wild-type (wt), deletion of the entire LEE region (del), insertion within the LEE (ins). Mice with a conventional microbiota SPF specific pathogen free. Other genomic changes listed in Supplemental table 1. e Depiction of a ~100,000 base-pair region of the C. rodentium genome containing the LEE pathogenicity island. Read depth from STAMP-CR69K inoculum and 5 clones isolated after 20 days passage in otherwise germ-free animals. Large deletions in 3 of 5 clones revealed by lack of specifically mapped reads in regions of up to 97,691 base-pairs. Non-specific reads map to multiple loci in the genome (primarily transposons).Full size imageMicrobiota disruption also impaired the capacity of mice to clear C. rodentium infection (Figs. 6a, 7a)12. Pathogen burden in the feces of germ-free mice did not decrease over time, in marked contrast to mice with an intact microbiota (specific pathogen free; SPF). Similarly, most cages of streptomycin-pretreated mice failed to clear the pathogen, with heterogeneity presumably caused by variation in the rebound of the microbiota after streptomycin treatment (Fig. 6a). Despite high fecal burdens, germ-free animals only exhibited mild diarrhea and did not lose weight for the 30 days of observation. These data indicate that the microbiota is the primary impediment to C. rodentium replication in the gastrointestinal tract, antagonizing the pathogen’s capacity to initiate a replicative niche and promoting its clearance.In germ-free and streptomycin pretreated animals the number of C. rodentium founders ceased to be fractionally related to dose; doses ranging 10,000-fold, from 106 to 1010 CFUs, all yielded a similar number of founders 5-days post-inoculation (Figs. 6b, 7b). These data suggest that there is an upper limit to the size of the C. rodentium founding population of ~105 on day 5 (i.e., a bottleneck caused by limited resources as illustrated in Fig. 2a). Furthermore, in the absence of a microbiota dependent bottleneck, the maximum size of the founding population continuously decreased for the 20 days of observation (Figs. 6c, 7c). Although there was no contraction in the C. rodentium burden following infection of germ-free animals, the maximum number of founders decreased from ~105 on day 5 to ~102 on day 20 (Fig. 7a, c). A decrease in diversity without a decrease in abundance suggests that C. rodentium adapts to the germ-free environment, introducing a new bottleneck caused by intra-pathogen competition.To test the hypothesis that C. rodentium evolved during colonization of germ-free animals, we sequenced the genomes of single C. rodentium colonies isolated from infected SPF or germ-free mice 5 or 20 days post inoculation. 5 days post-inoculation, the C. rodentium genomes isolated from SPF and germ-free mice were similar to the inoculum, with 6/10 colonies lacking detectable variations (Supplemental table 1). These data indicate that the initial contraction of the C. rodentium population observed during establishment of infection is not caused by selection of a genetically distinct subpopulation of the inoculum. By contrast, 20 days growth in the absence of a microbiota was always accompanied by changes in the C. rodentium genome. Notably, C. rodentium with structural variations in the LEE pathogenicity island became dominant in 4 out of 5 cages of infected germ-free animals (Fig. 7d, Supplemental table 1). These variations included large deletions of up to 97,691 bps (Fig. 7e, isolate from mouse F1) that eliminated the entire island, which is essential for colonization of SPF mice20. These genome alterations suggest that in the absence of a microbiota, a common mechanism for C. rodentium adaption to the host environment is to lose the LEE pathogenicity island. Thus, we conclude that competition among C. rodentium constricts the diversity of the population in the absence of a microbiota-dependent bottleneck, with organisms that lose the LEE virulence island outcompeting bacteria possessing the LEE. Additional mutations were also detected in the C. rodentium isolated on day 20 from germ-free animals, including in the galactonate operon, which have previously been observed in Escherichia coli colonizing microbiota depleted mice21 (Supplemental Table 1). Thus, there may be common evolutionary strategies for pathogenic and non-pathogenic bacteria to adapt to growth without competition in the host intestine.Collectively these experiments show that of the multiple host factors protecting against enteric infection, the microbiota is by far the most restrictive. Diminution of the microbiota markedly increases host susceptibility, permitting infection at almost any dose. In the absence of competition with the microbiota, a new slow-acting bottleneck constricted the C. rodentium population as the pathogen evolved increased fitness, notably through loss of the LEE pathogenicity island. More

Chala, B. & Hamde, F. Emerging and re-emerging vector-borne infectious diseases and the challenges for control: A review. Front. Public Health https://doi.org/10.3389/fpubh.2021.715759 (2021).Article 

Google Scholar 
Jongejan, F. & Uilenberg, G. The global importance of ticks. Parasitology 129, S3–S14 (2004).
Google Scholar 
Hornok, S., Kováts, D., Horváth, G., Kontschán, J. & Farkas, R. Checklist of the hard tick (Acari: Ixodidae) fauna of Hungary with emphasis on host-associations and the emergence of Rhipicephalus sanguineus. Exp. Appl. Acarol. 80, 311–328 (2020).
Google Scholar 
ECDC. Surveillance and disease data—Tick maps. https://www.ecdc.europa.eu/en/diseasevectors/surveillance-and-disease-data/tick-maps (2022). Accessed: 2022–09–02.Brites-Neto, J., Duarte, K. M. R. & Martins, T. F. Tick-borne infections in human and animal population worldwide. Vet. World 8, 301 (2015).
Google Scholar 
Hubálek, Z. Epidemiology of Lyme borreliosis. Lyme Borreliosis 37, 31–50 (2009).
Google Scholar 
Rizzoli, A. et al. Lyme borreliosis in Europe. Eurosurveillance 16, 19906 (2011).
Google Scholar 
Marques, A. R., Strle, F. & Wormser, G. P. Comparison of Lyme disease in the United States and Europe. Emerg. Infect. Dis. 27, 2017 (2021).
Google Scholar 
Jaenson, T. G., Jaenson, D. G., Eisen, L., Petersson, E. & Lindgren, E. Changes in the geographical distribution and abundance of the tick Ixodes ricinus during the past 30 years in Sweden. Parasit. Vectors 5, 1–15 (2012).
Google Scholar 
Medlock, J. M. et al. Driving forces for changes in geographical distribution of Ixodes ricinus ticks in Europe. Parasit. Vectors 6, 1–11 (2013).
Google Scholar 
Semenza, J. C. & Suk, J. E. Vector-borne diseases and climate change: a European perspective. FEMS Microbiol. Lett. https://doi.org/10.1093/femsle/fnx244 (2018).Article 

Google Scholar 
Sutherst, R. W. Global change and human vulnerability to vector-borne diseases. Clin. Microbiol. Rev. 17, 136–173 (2004).
Google Scholar 
Tabachnick, W. Challenges in predicting climate and environmental effects on vector-borne disease episystems in a changing world. J. Exp. Biol. 213, 946–954 (2010).CAS 

Google Scholar 
Sonenshine, D. E., Kocan, K. M. & de la Fuente, J. Tick control: Further thoughts on a research agenda. Trends Parasitol. 22, 550–551 (2006).
Google Scholar 
Willadsen, P. Tick control: Thoughts on a research agenda. Vet. Parasitol. 138, 161–168 (2006).
Google Scholar 
Goolsby, J. A. et al. Rationale for classical biological control of cattle fever ticks and proposed methods for field collection of natural enemies. Subtrop. Agric. Environ. 66, 7–15 (2016).
Google Scholar 
Singh, N. et al. Effect of immersion time on efficacy of entomopathogenic nematodes against engorged females of cattle fever tick, Rhipicephalus (= Boophilus) microplus. Southwest. Entomol. 43, 19–28 (2018).
Google Scholar 
Černý, J. et al. Management options for Ixodes ricinus-associated pathogens: A review of prevention strategies. Int. J. Environ. Res. Public Health 17, 1830 (2020).
Google Scholar 
Kapranas, A. et al. Encyrtid parasitoids of soft scale insects: Biology, behavior, and their use in biological control. Annu. Rev. Entomol. 60, 195–211 (2015).CAS 

Google Scholar 
Chirinos, D. T. & Kondo, T. Description and biological studies of a new species of Metaphycus Mercet, 1917 (Hymenoptera: Encyrtidae), a parasitoid of Capulinia linarosae Kondo & Gullan. Int. J. Insect Sci. 11, 1179543319857962 (2019).
Google Scholar 
Polaszek, A., Noyes, J. S., Russell, S. & Ramadan, M. M. Metaphycus macadamiae (Hymenoptera: Encyrtidae)–a biological control agent of macadamia felted coccid Acanthococcus ironsidei (Hemiptera: Eriococcidae) in Hawaii. PLoS ONE 15, e0230944 (2020).CAS 

Google Scholar 
Howard, L. Another chalcidoid parasite of a tick. Can. Entomol. 40, 239–241 (1908).
Google Scholar 
Hu, R., Hyland, K. & Oliver, J. A review on the use of Ixodiphagus wasps (Hymenoptera: Encyrtidae) as natural enemies for the control of ticks (Acari: Ixodidae). Syst. Appl. Acarol. 3, 19–28 (1998).
Google Scholar 
Collatz, J. et al. A hidden beneficial: Biology of the tick-wasp Ixodiphagus hookeri in Germany. J. Appl. Entomol. 135, 351–358 (2011).
Google Scholar 
Takasu, K. & Nakamura, S. Life history of the tick parasitoid Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Kenya. Biol. Control. 46, 114–121 (2008).
Google Scholar 
Collatz, J. et al. Being a parasitoid of parasites: host finding in the tick wasp Ixodiphagus hookeri by odours from mammals. Entomol. Experimentalis et Applicata 134, 131–137 (2010).
Google Scholar 
Krawczyk, A. I. et al. Tripartite interactions among Ixodiphagus hookeri, Ixodes ricinus and deer: Differential interference with transmission cycles of tick-borne pathogens. Pathogens 9, 339 (2020).
Google Scholar 
Plaire, D., Puaud, S., Marsolier-Kergoat, M.-C. & Elalouf, J.-M. Comparative analysis of the sensitivity of metagenomic sequencing and PCR to detect a biowarfare simulant (Bacillus atrophaeus) in soil samples. PLoS ONE 12, e0177112 (2017).
Google Scholar 
Wang, C.-X. et al. Comparison of broad-range polymerase chain reaction and metagenomic next-generation sequencing for the diagnosis of prosthetic joint infection. Int. J. Infect. Dis. 95, 8–12 (2020).CAS 

Google Scholar 
Tóth, A. G. et al. Ixodes ricinus tick bacteriome alterations based on a climatically representative survey in Hungary. bioRxiv (2022).Estrada-Peña, A., Mihalca, A. D. & Petney, T. N. Ticks of Europe and North Africa: A Guide to Species Identification (Springer, 2018).
Google Scholar 
Li, D., Liu, C.-M., Luo, R., Sadakane, K. & Lam, T.-W. MEGAHIT: An ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 31, 1674–1676 (2015).CAS 

Google Scholar 
Wood, D. E., Lu, J. & Langmead, B. Improved metagenomic analysis with Kraken 2. Genome Biol. 20, 1–13 (2019).
Google Scholar 
Pruitt, K. D., Tatusova, T. & Maglott, D. R. NCBI Reference Sequence (RefSeq): A curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 33, D501–D504 (2005).CAS 

Google Scholar 
NCBI Resource Coordinators. Database resources of the national center for biotechnology information. Nucleic Acids Res. 44, D7 (2016).
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).CAS 

Google Scholar 
Tennekes, M. tmap: Thematic maps in R. J. Stat. Softw. 84, 1–39 (2018).
Google Scholar 
Bodenhofer, U., Bonatesta, E., Horejš-Kainrath, C. & Hochreiter, S. msa: An R package for multiple sequence alignment. Bioinformatics 31, 3997–3999 (2015).CAS 

Google Scholar 
R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria (2022).Alfeev, N. & Klimas, Y. On the possibility of developing ichneumon flies, Hunterellus hookeri in climatic conditions of the USSR. Sovet. Vet. 15, 55 (1938).
Google Scholar 
Buczek, A., Buczek, W., Bartosik, K., Kulisz, J. & Stanko, M. Ixodiphagus hookeri wasps (Hymenoptera: Encyrtidae) in two sympatric tick species Ixodes ricinus and Haemaphysalis concinna (Ixodida: Ixodidae) in the Slovak Karst (Slovakia): Ecological and biological considerations. Sci. Rep. 11, 1–10 (2021).
Google Scholar 
Slovák, M. Finding of the endoparasitoid Ixodiphagus hookeri (Hymenoptera, Encyrtidae) in Haemaphysalis concinna ticks in Slovakia. Biol. Bratislava 58, 890–894 (2003).
Google Scholar 
Rehacek, J. & Kocianova, E. Attempt to infect Hunterellus hookeri Howard (Hymenoptera, Encyrtidae), an endoparasite of ticks, with Coxiella burnetti. Acta Virol. 36, 492–492 (1992).CAS 

Google Scholar 
Bohacsova, M., Mediannikov, O., Kazimirova, M., Raoult, D. & Sekeyova, Z. Arsenophonus nasoniae and Rickettsiae infection of Ixodes ricinus due to parasitic wasp Ixodiphagus hookeri. PLoS ONE 11, e0149950 (2016).
Google Scholar 
Boucek, Z. & Verny, V. A parasite of ticks, the chalcid Hunterellus hookeri in Czechoslovakia. Zool. Listy 3, 109–111 (1954).
Google Scholar 
Sormunen, J. J., Sippola, E., Kaunisto, K. M., Vesterinen, E. J. & Sääksjärvi, I. E. First evidence of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) parasitization in Finnish castor bean ticks (Ixodes ricinus). Exp. Appl. Acarol. 79, 395–404 (2019).CAS 

Google Scholar 
Doby, J. & van Laere, G. Hunterellus hookeri howard, 1907, Hymenoptère Chalcididae parasite de la tique Ixodes ricinus dans l’ouest et le centre de la France. Bull. de la Société française de parasitologie 11, 265–270 (1993).
Google Scholar 
Plantard, O. et al. Detection of Wolbachia in the tick Ixodes ricinus is due to the presence of the hymenoptera endoparasitoid Ixodiphagus hookeri. PLoS ONE 7, e30692 (2012).ADS 
CAS 

Google Scholar 
Japoshvili, G. New records of Encyrtids (Hymenoptera: Chalcidoidea: Encyrtidae) from Georgia, with description of seven new species. J. Asia-Pacific Entomol. 20, 866–877 (2017).
Google Scholar 
Walter, G. Beitrag zur Biologie der Schlupfwespe Hunterellus hookeri Howard (Hymenoptera: Encyrtidae) in Norddeutschland. Beitr. Naturkunde Niedersachsens 33, 129–133 (1980).
Google Scholar 
Ramos, R. A. N. et al. Occurrence of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Ixodes ricinus (Acari: Ixodidae) in Southern Italy. Ticks Tick-borne Dis. 6, 234–236 (2015).
Google Scholar 
Tijsse-Klasen, E., Braks, M., Scholte, E.-J. & Sprong, H. Parasites of vectors—Ixodiphagus hookeri and its Wolbachia symbionts in ticks in the Netherlands. Parasit. Vectors 4, 1–7 (2011).
Google Scholar 
Luu, L. et al. Bacterial pathogens and symbionts harboured by Ixodes ricinus ticks parasitising red squirrels in the United Kingdom. Pathogens 10, 458 (2021).CAS 

Google Scholar 
Pervomaisky, G. S. On the infestation of Ixodes persulcatus by Hunterellus hookeri How. (Hymenoptera). Zool. Zhurnal 22, 211–213 (1943).
Google Scholar 
Klyushkina, E. A parasite of the ixodid ticks, Hunterellus hookeri. How in the Crimea. Zool. Zh. 37, 1561–1563 (1958).
Google Scholar 
Gorman, M., Xu, R., Prakoso, D., Salvador, L. C. & Rajeev, S. Leptospira enrichment culture followed by ONT metagenomic sequencing allows better detection of Leptospira presence and diversity in water and soil samples. PLOS Neglected Trop. Dis. 16, e0010589 (2022).CAS 

Google Scholar 
Ranjan, R., Rani, A., Metwally, A., McGee, H. S. & Perkins, D. L. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem. Biophys. Res. Commun. 469, 967–977 (2016).CAS 

Google Scholar 
Laudadio, I. et al. Quantitative assessment of shotgun metagenomics and 16S rDNA amplicon sequencing in the study of human gut microbiome. OMICS 22, 248–254 (2018).CAS 

Google Scholar 
Munaf, H. et al. The first record of Hunterellus hookeri parasitizing Rhipicephalus sanguineus in Indonesia. Southeast Asian J. Trop. Medicine Public Heal. 7, 492 (1976).CAS 

Google Scholar 
Stafford, K. C. III., Denicola, A. J. & Kilpatrick, H. J. Reduced abundance of Ixodes scapularis (Acari: Ixodidae) and the tick parasitoid Ixodiphagus hookeri (Hymenoptera: Encyrtidae) with reduction of white-tailed deer. J. Med. Entomol. 40, 642–652 (2003).
Google Scholar 
Stafford, K. C. Jr., Denicola, A. J. & Magnarelli, L. A. Presence of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in two Connecticut populations of Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 33, 183–188 (1996).
Google Scholar 
Gillespie, J., Johnston, J., Cannone, J. & Gutell, R. Characteristics of the nuclear (18S, 5.8 S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): Structure, organization, and retrotransposable elements. Insect Mol. Biol. 15, 657–686 (2006).CAS 

Google Scholar 
Zhao, Y., Zhang, W.-Y., Wang, R.-L. & Niu, D.-L. Divergent domains of 28S ribosomal RNA gene: DNA barcodes for molecular classification and identification of mites. Parasit. Vectors 13, 1–12 (2020).
Google Scholar 
Larrousse, F., King, A. G. & Wolbach, S. The overwintering in Massachusetts of Ixodiphagus caucurtei. Science 67, 351–353 (1928).ADS 
CAS 

Google Scholar 
Smith, C. N. et al. Studies of parasites of the American dog tick. J. Econ. Entomol. https://doi.org/10.1093/jee/36.4.569 (1943).Article 

Google Scholar 
Hu, R., Hyland, K. E. & Mather, T. N. Occurrence and distribution in Rhode Island of Hunterellus hookeri (Hymenoptera: Encyrtidae), a wasp parasitoid of Ixodes dammini. J. Med. Entomol. 30, 277–280 (1993).CAS 

Google Scholar 
Scatolini, D. & Penteado-Dias, A. A fauna de Braconidae (hymenoptera) como bioindicadora do grau de preservação de duas localidades do Estado do Paraná. Revista Brasileira de Ecol. 1, 84–87 (1997).
Google Scholar 
Anderson, A. et al. The potential of parasitoid Hymenoptera as bioindicators of arthropod diversity in agricultural grasslands. J. Appl. Ecol. 48, 382–390 (2011).
Google Scholar  More