Ecology

Obtaining bacterial strains with varied resistance levelsWe experimentally evolved 24 replicate lineages of E. coli to tolerate increasing concentrations of chloramphenicol. By serially passaging bacterial cultures through 14 increasing chloramphenicol levels, we obtained 336 (24 lineages × 14 concentrations) populations of E. coli across a gradient of resistance levels (Fig. 2). The 24 replicate lineages enabled us to study the variability arising from the stochastic nature of mutation acquisition. We refer to these populations as “cultures” rather than “strains” due to the possible coexistence of multiple genotypes.Resistance incurs costs in both thermal tolerance and maximum growth rateWe measured growth rates of experimentally evolved E. coli cultures at three different temperatures: their historic temperature of 37 °C, and the novel temperatures of 32 °C and 42 °C. We hypothesized that growth rate costs of resistance would be larger in the novel temperatures, consistent with reduced thermal niche breadth.Overall, we found the growth rates decreased strongly with increasing antibiotic resistance (Fig. 3A). We then calculated relative growth rates for each lineage by dividing the growth rate at each timepoint by the growth rate of the culture at timepoint 1 (T1) at the appropriate temperature (e.g., all cultures at 32 °C were standardized by the ancestral growth rate at 32 °C). Analysis of these relative growth rates showed that there was both a fitness cost in maximum growth rate and a fitness cost in thermal niche breadth; the linear model showed a strong negative effect of increasing resistance on growth rate at 37 °C (F1, 974 = 988.2, p  More

Boyle, T. H. & Anderson, E. in Cacti: Biology and Uses (ed. Nobel, P. S.) 125–141 (Univ. California Press, 2002).Gibson, A. C. & Nobel, P. S. The Cactus Primer (Harvard Univ. Press, 1986).Bravo Hollis, H. & Sánchez Mejorada, H. Las Cactáceas de México (Univ. Nacional Autónoma de México, 1978).Goettsch, B. et al. High proportion of cactus species threatened with extinction. Nat. Plants 1, 15142 (2015).CAS 
PubMed 

Google Scholar 
Benavides, E., Breceda, A. & Anadón, J. D. Winners and losers in the predicted impact of climate change on cacti species in Baja California. Plant Ecol. 222, 29–44 (2021).
Google Scholar 
Nobel, P. S. Responses of some North American CAM plants to freezing temperatures and doubled CO2 concentrations: implications of global climate change for extending cultivation. J. Arid. Environ. 34, 187–196 (1996).
Google Scholar 
Reyes-García, C. & Andrade, J. L. Crassulacean acid metabolism under global climate change. N. Phytol. 181, 754–757 (2009).
Google Scholar 
Smith, S. D., Didden-Zopfy, B. & Nobel, P. S. High-temperature responses of North American cacti. Ecology 65, 643–651 (1984).
Google Scholar 
Larios, E., González, E. J., Rosen, P. C., Pate, A. & Holm, P. Population projections of an endangered cactus suggest little impact of climate change. Oecologia 192, 439–448 (2020).PubMed 

Google Scholar 
Esparza-Olguı́n, L., Valverde, T. & Vilchis-Anaya, E. Demographic analysis of a rare columnar cactus (Neobuxbaumia macrocephala) in the Tehuacan Valley, Mexico. Biol. Conserv. 103, 349–359 (2002).
Google Scholar 
Seal, C. E. et al. Thermal buffering capacity of the germination phenotype across the environmental envelope of the Cactaceae. Glob. Change Biol. 23, 5309–5317 (2017).
Google Scholar 
Huang, J., Yu, H., Guan, X., Wang, G. & Guo, R. Accelerated dryland expansion under climate change. Nat. Clim. Change 6, 166–171 (2016).
Google Scholar 
Gurvich, D. E. et al. Combined effect of water potential and temperature on seed germination and seedling development of cacti from a mesic Argentine ecosystem. Flora 227, 18–24 (2017).
Google Scholar 
Nuzhyna, N., Baglay, K., Golubenko, A. & Lushchak, O. Anatomically distinct representatives of Cactaceae Juss. family have different response to acute heat shock stress. Flora 242, 137–145 (2018).
Google Scholar 
Andrade, J. L. & Nobel, P. S. Microhabitats and water relations of epiphytic cacti and ferns in a lowland neotropical forest. Biotropica 29, 261–270 (1997).
Google Scholar 
Williams, D. G., Hultine, K. R. & Dettman, D. L. Functional trade-offs in succulent stems predict responses to climate change in columnar cacti. J. Exp. Bot. 65, 3405–3413 (2014).PubMed 

Google Scholar 
Aragón-Gastélum, J. L. et al. Induced climate change impairs photosynthetic performance in Echinocactus platyacanthus, an especially protected Mexican cactus species. Flora Morphol. Distrib. Funct. Ecol. Plants 209, 499–503 (2014).
Google Scholar 
Martorell, C., Montañana, D. M., Ureta, C. & Mandujano, M. C. Assessing the importance of multiple threats to an endangered globose cactus in Mexico: cattle grazing, looting and climate change. Biol. Conserv. 181, 73–81 (2015).
Google Scholar 
Dávila, P., Téllez, O. & Lira, R. Impact of climate change on the distribution of populations of an endemic Mexican columnar cactus in the Tehuacán-Cuicatlán Valley, Mexico. Plant Biosyst. 147, 376–386 (2013).
Google Scholar 
Conver, J. L., Foley, T., Winkler, D. E. & Swann, D. E. Demographic changes over >70 yr in a population of saguaro cacti (Carnegiea gigantea) in the northern Sonoran Desert. J. Arid. Environ. 139, 41–48 (2017).
Google Scholar 
Carrillo-Angeles, I. G., Suzán-Azpiri, H., Mandujano, M. C., Golubov, J. & Martínez-Ávalos, J. G. Niche breadth and the implications of climate change in the conservation of the genus Astrophytum (Cactaceae). J. Arid. Environ. 124, 310–317 (2016).
Google Scholar 
de Cavalcante, A. M. B. & de Duarte, A. S. Modeling the distribution of three cactus species of the Caatinga biome in future climate scenarios. Int. J. Ecol. Environ. Sci. 45, 191–203 (2019).
Google Scholar 
de Cavalcante, A. M. B., de Duarte, A. S. & Ometto, J. P. H. B. Modeling the potential distribution of Epiphyllum phyllanthus (L.) Haw. under future climate scenarios in the Caatinga biome. An. Acad. Bras. Cienc. 92, 351–358 (2020).
Google Scholar 
Tellez-Valdes, O. & DiVila-Aranda, P. Protected areas and climate change: a case study of the cacti in the Tehuacan-Cuicatlan biosphere reserve, Mexico. Conserv. Biol. 17, 846–853 (2003).
Google Scholar 
dos Santos Simões, S., Zappi, D., da Costa, G. M., de Oliveira, G. & Aona, L. Y. S. Spatial niche modelling of five endemic cacti from the Brazilian Caatinga: past, present and future. Austral Ecol. 45, 1–13 (2019).
Google Scholar 
Gorostiague, P., Sajama, J. & Ortega-Baes, P. Will climate change cause spatial mismatch between plants and their pollinators? A test using Andean cactus species. Biol. Conserv. 226, 247–255 (2018).
Google Scholar 
Butler, C. J., Wheeler, E. A. & Stabler, L. B. Distribution of the threatened lace hedgehog cactus (Echinocereus reichenbachii) under various climate change scenarios. J. Torre. Bot. Soc. 139, 46–55 (2012).
Google Scholar 
Johnson, C. N. Species extinction and the relationship between distribution and abundance. Nature 394, 272–274 (1998).CAS 

Google Scholar 
Thuiller, W., Lavorel, S. & Araújo, M. B. Niche properties and geographical extent as predictors of species sensitivity to climate change. Glob. Ecol. Biogeogr. 14, 347–357 (2005).
Google Scholar 
Enquist, B. J. Cyberinfrastructure for an integrated botanical information network to investigate the ecological impacts of global climate change on plant biodiversity. Preprint at PeerJ https://doi.org/10.7287/peerj.preprints.2615v2 (2016).Buisson, L., Thuiller, W., Casajus, N., Lek, S. & Grenouillet, G. Uncertainty in ensemble forecasting of species distribution. Glob. Change Biol. 16, 1145–1157 (2010).
Google Scholar 
Thuiller, W., Guéguen, M., Renaud, J., Karger, D. N. & Zimmermann, N. E. Uncertainty in ensembles of global biodiversity scenarios. Nat. Commun. 10, 1446 (2019).PubMed 
PubMed Central 

Google Scholar 
Goettsch, B., Durán, A. P. & Gaston, K. J. Global gap analysis of cactus species and priority sites for their conservation. Conserv. Biol. 33, 369–376 (2018).PubMed 

Google Scholar 
Maitner, B. S. et al. The bien R package: A tool to access the Botanical Information and Ecology Network (BIEN) database. Methods Ecol. Evol. 9, 373–379 (2018).
Google Scholar 
Karger, D. N. et al. Climatologies at high resolution for the Earth’s land surface areas. Sci. Data 4, 170122 (2017).PubMed 
PubMed Central 

Google Scholar 
Sanderson, B. M., Knutti, R. & Caldwell, P. A representative democracy to reduce interdependency in a multimodel ensemble. J. Clim. 28, 5171–5194 (2015).
Google Scholar 
Brodzik, M. J., Billingsley, B., Haran, T., Raup, B. & Savoie, M. H. EASE-Grid 2.0: Incremental but significant improvements for Earth-gridded data sets. ISPRS Int. J. Geo-Inf. 1, 32–45 (2012).
Google Scholar 
Venter, O. et al. Global terrestrial human footprint maps for 1993 and 2009. Sci. Data 3, 160067 (2016).PubMed 
PubMed Central 

Google Scholar 
Phillips, S. maxnet: Fitting ‘maxent’ species distribution models with ‘glmnet’. R package version 0.1.4. https://CRAN.R-project.org/package=maxnet (2017).Friedman, J., Hastie, T. & Tibshirani, R. Regularization paths for generalized linear models via coordinate descent. J. Stat. Softw. 33, 1–22 (2010).PubMed 
PubMed Central 

Google Scholar 
Dormann, C. F. et al. Collinearity: a review of methods to deal with it and a simulation study evaluating their performance. Ecography 36, 27–46 (2013).
Google Scholar 
Franklin, S. B., Gibson, D. J., Robertson, P. A., Pohlmann, J. T. & Fralish, J. S. Parallel analysis: a method for determining significant principal components. J. Veg. Sci. 6, 99–106 (1995).
Google Scholar 
Roberts, D. R. et al. Cross-validation strategies for data with temporal, spatial, hierarchical, or phylogenetic structure. Ecography 40, 913–929 (2017).
Google Scholar 
Merow, C., Smith, M. J. & Silander, J. A. A practical guide to MaxEnt for modeling species’ distributions: what it does, and why inputs and settings matter. Ecography 36, 1058–1069 (2013).
Google Scholar 
Allouche, O., Tsoar, A. & Kadmon, R. Assessing the accuracy of species distribution models: prevalence, kappa and the true skill statistic (TSS). J. Appl. Ecol. 43, 1223–1232 (2006).
Google Scholar 
Calabrese, J. M., Certain, G., Kraan, C. & Dormann, C. F. Stacking species distribution models and adjusting bias by linking them to macroecological models. Glob. Ecol. Biogeogr. 23, 99–112 (2014).
Google Scholar 
R Core Team R: A Language and Environment for Statistical Computing Version 3.6.0 (R Foundation for Statistical Computing, 2019). https://www.R-project.org/ More

Systematic palaeontology
Tetrapoda Jaekel, 1909 fide Sues20
Family undesignated
Termonerpeton makrydactylus gen. et sp. nov. (Fig. 1)Fig. 1: Termonerpeton makrydactylus gen. et sp. nov. holotype UMZC 2019.1.a Specimen photograph. b Interpretive drawing. Scale bars 10 mm. Abbreviations: acet acetabulum, fem femur, fib fibula, ha haemal arch, ic intercentrum, l left, na neural arch, phal phalanx, piliac p post-iliac process, plc pleurocentrum, r right, sac rib sacral rib, tib tibia.Full size image

EtymologyGenus: from τέρμωυ (térmon) meaning boundary and ερπετό (erpetó) meaning ‘crawler’, referring to the field boundary walls near the East Kirkton quarry where the late Stan Wood initially discovered fossils from the East Kirkton Limestone and from where the type specimen may have been collected; species: from μακρύς (makrýs) meaning ‘elongate’ and δάχτυλο (dáchtylo; more precisely, δάχτυλο ποδιού, dáchtylo podioú) meaning ‘toe’, referring to the very long pedal digit IV.HolotypeUniversity of Cambridge Museum of Zoology (UMZC) 2019.1. A partial tetrapod postcranium, preserving both pelves, a femur, fibula, tibia, and an almost complete but disarticulated pes. Closely associated with the appendicular elements are dorsally open hoop-shaped centra, a few neural and haemal arches, curved ribs, and a section of articulated gastralia.Locality and horizonEast Kirkton quarry, near Bathgate, Scotland, UK. East Kirkton Limestone, Bathgate Hills Volcanic Formation. Exact horizon is unknown. Brigantian, Viséan, early Carboniferous (=Mississippian)21.Differential diagnosisPossible autapomorphies: ilium with drawn-out, flat, blade-like dorsal process; very large, stout, and elongate metatarsal IV, greatly exceeding the length of metatarsals III and V (~30% or more). Possible tetrapod synapomorphies among post-Devonian taxa: distinct interepipodial space between tibia and fibula; well-ossified tarsus comprising tibiale, fibulare, intermedium, four centralia, and five distal tarsals. Possible amniote synapomorphies, but often showing reversed polarity in several stem- and crown amniote taxa: presumed pedal phalangeal formula 23454; robust and long pedal digit IV; enlarged intermedium and fibulare, together occupying more than half of proximal moiety of tarsus; curved ribs. Characters of uncertain polarity (also present in Caerorhachis): elongate, slender, and posterodorsally oblique post-iliac process; short puboischiadic plate with almost vertical anterior margin; stout femur with poorly pronounced waisting along the shaft, longer than puboischiadic plate; hoop-shaped centra.Attributed specimenNational Museums Scotland (NMS) G.1992.22.1. An articulated, partially complete, large tetrapod pes, preserving a nearly complete array of tarsals, all metatarsals, and the proximal phalanges of digits I–III. Unit 82, East Kirkton Limestone, East Kirkton quarry, near Bathgate, Scotland, UK.
Specimen description
Appendicular skeletonMost of the description is based upon the holotype. Both pelves are preserved, one mainly as a natural mould. The puboichiadic plates are short and deep, with an almost vertical anterior margin to the pubis (Fig. 1). In one, the surface of the puboischiadic plate is strongly convex, in the other it is strongly concave. The concave plate may belong to the left pelvis, with the concavity indicating the acetabulum. Both iliac processes of the presumed right ilium are overlain by a neural arch and part of the femur and cannot be seen. The presumed left ilium shows a long, posteriorly pointing post-iliac process that extends as far backward as the posterior edge of the ischium. It retains the proximal, stump-like portion of a dorsal iliac process, continued distally in natural mould as a mediolaterally flattened and blade-like structure. Both processes sit above a short iliac neck. The dorsal iliac process is proportionally longer than in other tetrapods and its knife-like appearance is unique. The angle between the two processes is much more acute than in most other tetrapods, and the nearest comparison is with the divided iliac process of the microsaurs Tuditanus and Ricnodon22 which, however, could merely represent a bifid post-iliac process. Two gaps in ossification are taken as evidence of an ilio-ischiadic suture half-way down the posterior margin on the left pelvis and an ilio-pubic suture halfway down the anterior margin of the right pelvis (Fig. 1). There is no evidence of a puboischiadic suture, although a shallow depression along the ventral margin of the left puboischiadic plate probably marks the junction between pubis and ischium. The complete left puboischiadic plate is 20 mm deep behind the ilium and 30 mm long, with the pubis contributing about one-third of its length and the ischium the remaining two thirds. The anterior margin of the pubis is almost vertical. The dorsal margin of the ischium is shallowly convex for half its length before extending posteroventrally to meet the upturned posterior extremity of the ischium’s ventral margin. There is no evidence as to the angle at which the two pelvic plates met at the symphysis, which would affect the position of the acetabulum relative to the substrate, and thus the effective resting posture of the hindlimb.The left femur is at least 39 mm in length, and longer than the puboischiadic plate. The entire bone is crushed, and its distal end lies partly beneath one of the pelvic halves and a neural arch so that its features cannot easily be made out. A possible intercondylar groove may be present distally, and the extensor surface of its proximal extremity appears to show a subcentral depression. The femur itself is robust with little waisting at mid-shaft. A small internal trochanter lies near its proximal end. The left fibula is approximately 26 mm long along its lateral margin. Its proximal end is narrow and grooved. Its broad and strongly flared distal end suggests a broad contact with the tarsus. The medial turn of the distal end indicates a large interepipodial space. The left tibia is about 20 mm long, slender, and shallowly waisted at mid-shaft. It is not clear which end is proximal and which distal, although probably the proximal is the broader. The tibia is probably more than half the length of the femur. Based upon the femur and tibia lengths, and omitting the ankle and pes, the above figures indicate a total stylopod-zeugopod length of about 65 mm, assuming a fully extended limb.Most of the morphology of the left pes is preserved, showing many well-ossified tarsal bones (Fig. 2). Several of these, including possible distal tarsals II and III lie more or less in anatomical continuity relative to metatarsals II and III, respectively. Other tarsal elements, including possible fibulare, tibiale, centralia, and distal tarsals, are illustrated in Fig. 2. Metatarsal IV lies in anatomical position relative to metatarsals II and III and, at 7 mm in length, it is significantly larger than the latter. The presumed first phalanx of pedal digit IV lies close to metatarsal IV, at an angle of nearly 90° to the latter. It is long and slender, indicating an unusually elongate fourth pedal digit.Fig. 2: Termonerpeton makrydactylus gen. et sp. nov. left hindlimb of UMZC 2019.1.a Specimen photograph, showing close-up view of hindlimb skeleton, b Interpretive drawing, with centralia, distal tarsals, and metatarsals indicated by red, blue, and black Roman numerals, respectively, c Interpretive drawing with dashed lines connecting elements of individual digits, d Reconstruction of left tibia, fibula and pes. Scale bars 10 mm. Abbreviations: interm intermedium, tib tibialia.Full size imageAn array of about 12 phalanges is preserved. They are all disrupted but occur in proximity to one another and, like the first phalanx of pedal digit IV, also mainly lie at right angles to metatarsals III and IV. An additional, acutely angled pointed ungual phalanx, possibly associated with digit II, is also visible. A further two phalanges have been displaced and rest along the anterior edge of the left pelvis. In total, we were, therefore, able to identify 15 elements. The preservation of the pes suggests it was strongly flexed either at death or from tissue shrinkage thereafter. An isolated metatarsal, presumably from the other, missing foot, lies some distance away near the edge of the block. Together, the pedal elements suggest a relatively large foot.A second specimen, NMS G.1992.22.1 (Fig. 3), is represented by an isolated pes. It may belong to Termonerpeton, although it is from a much larger individual. It shows five metatarsals of which the fourth is much longer and more robust than the other four and about twice as long as that of the holotype, while metatarsal V is the smallest. There are three phalanges, plus five distal tarsals. A D-shaped element closely associated with three centralia could be either a fibulare, a displaced intermedium, or centrale IV.Fig. 3: Termonerpeton makrydactylus gen. et sp. nov partial pes, attributed specimen NMS G.1992.22.1.a Specimen photograph, b with centralia, distal tarsals, and metatarsals indicated by red, blue, and black Roman numerals, respectively. Scale bars 10 mm.Full size imageAxial skeletonWhere visible, neural arches have short neural spines and prominent zygapophyses, but their shape is hard to assess as none is well preserved. The element overlying part of the right pelvis and the femur is 7 mm high in total. Numerous dorsally open, hoop-shaped centra about 5 mm in diameter are visible, as well as a few small, oval, shallowly curved elements (Fig. 1). Without further evidence, it is uncertain which of these elements are intercentra and which pleurocentra, though we assume that the larger elements are pleurocentra. The preserved ribs are slender and curved, and include trunk ribs, a possible presacral rib, a possible sacral rib, and a possible postsacral rib. This is long but more or less straight. A bone situated among a cluster of centra, somewhat distant from the other tarsal bones, was originally interpreted by us as a possible fibulare, similar to the fibulare in Proterogyrinus23. However, it might also be interpreted as a sacral rib. If so, its morphology is unique. It is short and widens distally into a fan-shaped structure but does not appear to have a bifid proximal end, unlike the sacral rib in Proterogyrinus23. Three haemal arches are present, one still attached to its half-hoop centrum, a second slightly longer, and a third very short and presumably from a more posterior region of the tail.ComparisonsThe exceptional preservation of tetrapods from the East Kirkton Limestone provides a unique opportunity to study portions of the skeletal anatomy that are otherwise poorly preserved or absent among Mississippian tetrapods. In particular, hindlimbs with a complete or near-complete array of tarsal elements and digits are notably rare. The unusual construction of the pes of Termonerpeton prompted us to examine the hindlimb morphology of six other East Kirkton tetrapods (Fig. 4a–g) alongside a selection of additional, mostly Carboniferous taxa (Fig. 4h–n). We focus on epipodials, tarsi, phalangeal formulae and digit length and proportions. To facilitate visual inspection of these elements, all hindlimbs are drawn to a common tibial length, except for the stem diapsid Petrolacosaurus, in which the epipodials are greatly elongate.Fig. 4: Comparison of the left tibia, fibula, tarsus, and digits of early tetrapods.a Balanerpeton after 2, b Eucritta after 12, c Eldeceeon after 6, d Silvanerpeton after 4, e Westlothiana after 7, f Kirktonecta original, see 15 (the grey area marks the estimated position and extent of the tarsus), g Termonerpeton, h Pederpes after 24, i, Greererpeton after 27, j Caerorhachis after 31, k Archeria after 30, l Hylonomus after 28, m Tuditanus after 22, n Petrolacosaurus after 29. Drawn to the same tibial length apart from n. Scale bars 10 mm.Full size imageIn terms of pes size relative to the tibia, the East Kirkton taxa Balanerpeton, Eucritta, and Silvanerpeton (Fig. 4a, b, d) are similarly proportioned. In contrast, Eldeceeon and Westlothiana (Fig. 4c, e) exhibit somewhat larger pedes. Kirktonecta has proportionally the largest pedes of all (Fig. 4f). Termonerpeton (Fig. 4g) has a pes of similar size to the first three taxa except that digit IV is relatively much longer than in any of the others, with an exceptionally large metatarsal IV. In all those taxa in which digit IV is fully preserved, it is the longest, especially in Eldeceeon and Kirktonecta, but in none does it approach in size and proportions that of Termonerpeton. The illustrated limbs also differ from one another in the degree of ossification of the tarsal bones. Most taxa except Eucritta have some indication of ossified tarsal elements, and some of them, including Balanerpeton and Silvanerpeton, show a complete or almost complete tarsal set. Kirktonecta does have an ossified tarsus, but specimen preservation does not allow us to identify individual elements. The phalangeal count, where known, also varies: 22343 in Balanerpeton2; 223?? in Eucritta12; 23455 in Silvanerpeton4; 23454 in Eldeceeon6, Kirktonecta15, Termonerpeton, and Westlothiana7.In addition, we compared the pedes of East Kirkton tetrapods with those of seven other taxa (Fig. 4h–n): one earlier, Pederpes24; one almost contemporary, Caerorhachis25; four later Carboniferous, Greererpeton26, Hylonomus27, Tuditanus22, and Petrolacosaurus28; and one early Permian, Archeria29. Of these, Greererpeton has relatively the smallest pes. In most, digit IV is the longest, though in Pederpes and Caerorhachis it is incomplete. The pes of Caerorhachis was originally restored with only three phalanges in digit IV30. This is probably incorrect and would be unusual in Carboniferous tetrapods. The pes of the anthracosaur Archeria was originally reconstructed with digit V as the longest29, but again this is unusual among later Carboniferous and early Permian tetrapods and we suspect that digits IV and V have been transposed, and Romer himself expressed doubt about this reconstruction29. In either case, the phalangeal formula of Archeria is similar to that of the East Kirkton anthracosaur Silvanerpeton, as 23455.Among Carboniferous tetrapods, temnospondyls such as Balanerpeton and colosteids such as Greererpeton show a digit IV that is somewhat longer than the others, but metatarsal IV is very similar in length and breadth to the adjacent metatarsals. In anthracosaurs, digit IV is the longest, but again metatarsal IV is not significantly broader than adjacent metatarsals. This is also the case in the early amniote Hylonomus and the microsaur Tuditanus. Among the taxa illustrated here, Termonerpeton shows a strikingly similar pes to that of the Late Pennsylvanian araeoscelidian diapsid Petrolacosaurus (Fig. 4n). In both, metatarsal IV is significantly longer and stouter than others and forms part of a similarly long digit IV. In early amniotes, an elongate digit IV coupled with an elongate metatarsal IV is a common occurrence in other taxa, such as protothyridids (e.g. Anthracodromeus31), basal araeoscelidians (e.g. Spinoaequalis32), younginids (e.g. Youngina33), saurians33, and basal synapsids (e.g. Heleosaurus34,35,36), among others.Based upon available evidence, an elongate digit IV is likely to be the plesiomorphic condition for crown amniotes, being present in Hylonomus, Paleothyris, and Petrolacosaurus (Fig. 4l, n), and shortening of this digit certainly represents a derived feature. In later crown amniotes, the conditions vary, with larger, heavier-bodied tetrapods such as dicynodonts and diadectids having generally shorter toes and adopting a more clearly plantigrade posture. An elongate metatarsal IV and associated digit, however, are not universal among Palaeozoic amniotes, and modifications of these conditions occur repeatedly across clades. For instance, in the eureptile captorhinid Eocaptorhinus, digit IV is also the longest, but the length of metatarsal IV does not greatly exceed that of other metatarsals37. The same is true of some early Permian clades, including seymouriamorphs (e.g. Seymouria38; Discosauriscus39), and diadectids (e.g. Diadectes40), although in the diadectomorph Orobates digit III is a little longer than digit IV41. Among synapsids, dicynodonts such as Diictodon42 and caseids43, to name a few, have five pedal digits of approximately uniform length.We further point out that, while digit IV attains a certain degree of elongation in other early tetrapod groups, such as temnospondyls, in none of them do the relative proportions of this digit (where known) compare to those of several stem and crown amniotes (Fig. 4).Phylogenetic relationshipsThe results of various phylogenetic analyses lend some support to the interpretation of Termonerpeton as a stem amniote, despite its uncertain placement in the unweighted character parsimony analysis (Fig. 5a). In the latter analysis, Termonerpeton appears in a polytomous node alongside baphetids (Eucritta; Baphetes; Megalocephalus), temnospondyls (Balanerpeton; Dendrysekos), the anthracosauroids Eldeceeon and Silvanerpeton, and the problematic Caerorhachis. In all other analyses—implied weights, reweighted characters, and Bayesian—Termonerpeton is placed on the amniote stem group, albeit in different positions, among a diverse array of ‘reptiliomorph’ clades and grades. In the implied weights analysis (Fig. 5b), Termonerpeton, Silvanerpeton, and Eldeceeon form a monophyletic group branching crownward of chroniosaurs plus anthracosaurs and anti-crownward of paraphyletic gephyrostegids. In the reweighted analysis (Fig. 5c), Termonerpeton and Caerorhachis appear as successive sister taxa, in that order, to monophyletic anthracosaurs. In the Bayesian analysis (Fig. 5d), the amniote total group receives moderate support with a credibility value (c.v.) of 76 with Caerorhachis as the most plesiomorphic stem amniote. Crownward of Caerorhachis is a polytomy with low support (c.v. = 59) that subtends Termonerpeton, a clade consisting of Eldeceeon plus Silvanerpeton, a clade of anthracosaurs, and a clade that includes all remaining taxa. In crownward succession, these taxa include chroniosaurs, gephyrostegids, seymouriamorphs, Solenodonsaurus, and Westlothiana as successive sister groups to a strongly supported (c.v. = 100) clade containing diadectomorphs, synapsids, and eureptiles. Although eureptile monophyly is not retrieved, strong support (c.v. = 100) is given to the branch subtending diadectomorphs plus synapsids44.Fig. 5: Results of phylogenetic analyses.a Strict consensus of 120 shortest trees from unweighted analysis (tree length = 1286 steps, ensemble consistency index C.I. = 0.2738 without uninformative characters, ensemble retention index R.I. = 0.5768), b Single tree from implied weights analysis (tree length = 1298 steps, Goloboff fit = −202.59266, C.I. = 0.2712, R.I. = 0.5713), c Single tree from reweighted analysis (tree length = 212,68965 steps, C.I. = 0.4755, R.I. = 0.774), d Bayesian topology with branches reporting credibility values.Full size image More

Species used in the experimentsMilnesium inceptum32 (Fig. 1A, a picture taken using Olympus BX41 Phase Contrast light Microscope associated with Olympus SC50 digital camera) is an obligatory predatory species with the body length ranging from 326 to 848 μm. It feeds on rotifers, nematodes and other tardigrades and lays smooth eggs in exuviae. To stay active, M. inceptum needs a thin water film around its body14. The species inhabits places exposed to shorter and longer periods of drying i.e. frequently drying mosses growing on cement walls32. Till now it was reported in Poland, Germany, Japan, Switzerland and Bulgaria32. At the same time, it is a perfect organism for our research because (1) it is large and easy to observe, (2) it tolerates frequent periods of entering and leaving anhydrobiosis, (3) it easily creates a tun stage. Milnesium inceptum for experimental purposes were acquired from a moss sample from a cement wall in Poznań, Poland (52°24′15″N, 16°53′18″E). The extraction of tardigrades was conducted under stereomicroscope (Olympus SZ51) using standard methods33. Then specimens, further used in our experiments, have been cultured based on protocol proposed by Roszkowska et al.34. Only fully active, adult specimens were selected for the experiments.Figure 1Model animals used in experiments: (A) Milnesium inceptum; insert shows tardigrade in the tun state; (B) Cepaea nemoralis in its natural environment; (C) a tardigrade that appeared on moss surface during in vivo observation of rehydrated moss cushion (red arrow). Figures were assembled in Corel Photo-Paint 2017 (http://www.corel.com).Full size imageCepaea nemoralis35 (Fig. 1B, a picture taken using Motorola g(9), Camera version 7.3.63.53-whitney) is a stylommatophoran European land snail species, which is widespread and common throughout the continent36. The average maximum shell diameter is 20 to 22 mm37. It feeds on plant materials available, yet has a strong preference for dead and senescent herbs38. C. nemoralis occurs in variable habitats (frequently in synanthropic ones) such as forests, meadows, gardens, near shrubs or dunes36.The period of its activity falls on the growing season; it usually comes out of the shell and crawls when the air humidity reaches 70% or more, independently from solar radiation and air temperature28. The species is a good model for our study due to its: (1) large size compared to tardigrades, and (2) co-occurrence with M. inceptum in natural environments. Individuals of C. nemoralis were harvested from anthropogenic environment: gardens adjacent to detached houses (52°25′28″N, 16°46′52″E). Snails were collected from plants, cement walls and ground surfaces. After collection, all C. nemoralis specimens were washed-up and placed in 30 L (480 × 360 × 252 mm) transparent plastic box with mesh covering for ventilation. Soil and rocks were placed in the box allowing to maintain a moist shelter for snails, and a sepia was used as a source of a calcium. Animals were fed with lettuce, cabbage and nettle twice a week and sprinkled with water to stimulate their activity. Box containing snails was kept in a rearing room, at 17 °C in 12:12 photoperiod. Snails were kept in the box for 1.5 months prior to the experiments. For the experiments we used only adult animals. The snails were checked under Olympus SZX7 stereomicroscope prior to the experiment to ensure they were free of tardigrades.Pilot studiesDoes the tardigrades’ distribution within a moss cushion enable tardigrade-snail contact?To check whether tardigrades may come into a close encounter with the snail in the natural environment (which would be impossible if the tardigrades were only present in the lower layers of the moss), we investigated the distribution of water bears within moss cushions. The observations were performed for 6 samples of dried moss cushions (ca. 1 cm high and 3 cm in diameter). The moss containing M. inceptum specimens, was collected from a concrete wall in Poznań, Poland (52°24′15″N, 16°53′18″E), the same from which tardigrades were initially collected for the culturing purposes. Three moss cushions were rehydrated, and left for 3 h followed by further observation to check whether tardigrades may actively move across the moss cushion. On the remaining three moss samples, a horizontal cut was made through the center of the moss cushion to check in which layer tardigrade tuns are present while the moss remains dry. The extraction of tardigrades from separated layers was conducted under stereomicroscope (Olympus SZ51) using standard methods33.Within the dry moss cushions tardigrades were present in both the upper and lower moss layers. We did not observe any difference in the number of individuals of M. inceptum that would be dependent on the moss layer. A total of 353 tardigrades were extracted from one moss cushion (dry weight of moss = 0.332 g), what gives the density of tardigrades per 1 g of dry moss sample equal to 1063 specimens. The observation of rehydrated moss cushions conducted in vivo using Olympus SZX16 stereomicroscope associated with Olympus DP74 digital camera and cellSens software revealed that single active tardigrades may also appear on the moss surface (Fig. 1C, red arrow). Therefore, observed in the pilot studies tardigrades distribution within the moss cushion enables tardigrade-snail contact.Is it possible for a tardigrade to take a snail ride?The initial observations were carried out for snails and tardigrades to check whenever a tardigrade may be transferred by a snail. In total, 10 snails and 20 active tardigrades were used. Two variants of Petri dishes (ø 90 mm) were prepared: (1) with smooth and (2) scratched bottom, to avoid and allow tardigrade attachment to the bottom of the dish, respectively. We repeated the observation five times per option. For each single observation we used one snail and two tardigrades.Snails and tardigrades were split equally between the pilot’s experimental options (in total 5 snails and 10 tardigrades per option). We checked whether tardigrades may be transferred by snails by putting tardigrades in the drop of water in the center of a Petri dish and releasing an active snail to crawl through the drop. In total, in the case of the smooth-bottom option, three tardigrades glued to the snail’s body within which two were moved to a distance up to a few centimeters. The third one fixed to a snail’s leg and had a potential to be transferred to a greater distance. In the case of the dishes with the scratched bottom, we did not notice any transfer. Tardigrades were attached tightly to the dishes’ bottom and remained unmoved after the snail had passed through them. Therefore, the observation in the pilot study confirmed that tardigrades may stick to snails’ body and be transferred by a gastropod at least when the substratum (bottom of the dish) is smooth.Experimental design
Experiment 1. Do snails have a significant effect on tardigrade dispersion that depends on the substrate type?As the laboratory environment offers limited possibilities to reflect natural conditions, we aimed to create an environment similar to the natural one by eliminating as many artificial elements as possible and, at the same time, enabling observation and data collection. To imitate a natural microhabitat of water bears we used a piece of moss as a substrate. Moss is a natural shelter and a hunting space for these animals, and a gripping surface that prevents them from being easily carried away by a stream of water or wind. The moss Vesicularia dubyana39 used in the experiment was purchased in an aquarium shop and was derived from an in vitro culture. It was checked under Olympus SZX7 stereomicroscope prior to the experiment to ensure it was free of tardigrades. For experimental purposes we used plastic ventilated boxes with dimensions 950 mm × 950 mm × 600 mm, tightly closed with a plastic lid. The bottom of each box was scratched with sandpaper in order to (1) imitate a rough surface of a concrete wall to which mosses are attached in the natural environment; (2) allow tardigrade locomotion. At the same time, moss and (unfortunately) plastic elements are quite common surroundings of C. nemoralis frequently found in anthropogenic habitats36.Using transparent, non-toxic aquarium silicone, a square with a side length of 3 cm and a height of 0.5 cm was mounted on the bottom of the box. Before starting the experiment, the tightness of the square silicone barrier was checked by pouring 2.5 ml of water inside and leaving the boxes for observation for 24 h. After this time, all silicone squares turned out to be impermeable to water.Boxes for each of the experimental option, namely: (A) control (further in the text referred as C), (B) tardigrades + snail (referred as TS), and (C) tardigrades + snail + moss (referred as TSM, see Fig. 2), were prepared in a following way: 2.5 ml of water was added to the scratched bottom of the box inside the silicone square and 7.5 ml to the area outside of the silicone square to enable survival and active locomotion of tardigrades on both sides of the silicone barrier. Then, 10 active individuals of M. inceptum taken from the culture were transferred to the center of the silicone square. It was repeated for 90 boxes (30 boxes per each C, TS and TSM option). Therefore we used 300 tardigrades per each experimental option which gives 900 tardigrades in total for all experimental options. In case of 30 boxes with TSM option, a piece of moss (ca. 2.5 cm in diameter) was added. It was situated in the center of the silicone square, just after the tardigrades were placed at the boxes in order to isolate tardigrades from the snail during the experiment.Figure 2Graphical representation of three designed experimental options of the experiment 1. (A) 10 tardigrades in the silicone square (control (C)); (B) 10 tardigrades in the silicone square and one snail placed in the box (tardigrades + snail (TS)); (C) 10 tardigrades in the silicone square, one snail placed in the box and additional piece of the moss added as a barrier between tardigrades and snail (tardigrades + snail + moss (TSM)). Figures were assembled in Corel Photo-Paint 2017 (http://www.corel.com).Full size imageFinally, in the boxes targeted for TS and TSM experimental options, one adult and active individual of C. nemoralis snail was placed in each box outside the silicone square. In total, 60 snails were used (30 individuals per experimental option).The boxes were then placed in the rearing room (17 °C, 80% of humidity, photoperiod 12:12) for 72 h. After this time, the number of tardigrades inside and outside the silicone square was counted (both: live and dead) separately for each box, using Olympus SZX7 stereomicroscope.Experiment 2. Effect of the snail’s mucus on tardigrade recovery to active life after anhydrobiosis
Milnesium inceptum anhydrobiosis protocolOnly fully active, adult specimens of medium body length were selected for the experiment. The animals were transferred to ø 3.5 cm vented Petri-dishes with bottom scratched by sandpaper to allow tardigrade locomotion. Five tardigrade individuals were placed to each Petri dish together with 450 µl of water and then dehydrated. In total, 16 Petri dishes with 5 tardigrades on each were prepared. Dehydration process lasted 72 h and was performed in the Q-Cell incubator (40–50% RH, 20 °C, darkness). After that time tardigrade tuns were kept under the abovementioned conditions for 7 days.Impact of the snail’s mucus on tardigrade tunsAfter 7 days of anhydrobiosis, one individual of C. nemoralis was transferred to each dish with tardigrade tuns and was left there for 1 min allowing the snail to actively crawl over the tuns. 30 min after the snail was removed from the dish, tardigrade tuns were observed under the Olympus SZX7 stereomicroscope for any animal movements. Then, all covered and vented dishes were left in the Q-Cell incubator overnight. After 24 h, the dried tuns were rehydrated by adding 3 ml of water to each Petri dish to check whether snail’s mucus affected mortality rates of tardigrades. After 3 and 24 h following rehydration tardigrade tuns were observed for any animal movements. Pictures of tuns were taken using Olympus SZ61 stereomicroscope associated with Olympus UC30 camera (Fig. 3). As reference data on the rehydration of the M. inceptum tuns free of the snail’s mucus, we used the data from Roszkowska et al.20 who tested anhydrobiosis survivability of above-mentioned species. Individuals used for the tuns preparation in the control option were collected from the same laboratory breeding stock, and prepared at the same laboratory conditions as those used in our experiments20.Figure 3Milnesium inceptum tuns: (A,B) before contact with snail mucus; (C,D) coated with wet snail mucus; (E,F) coated with dry snail mucus. Figures were assembled in Corel Photo-Paint 2017 (http://www.corel.com).Full size imageStatistical analysesThe number of tardigrades relocated in each experimental option (C, TS and TSM) was compared with a one-way ANOVA randomized version using RundomPro 3.14 software40. We used non-parametric methods because of the lack of normality. Differences were considered significant at p  More

Bowman, D., Williamson, G. J., Gibson, R. K., Bradstock, R. A. & Keenan, R. J. The severity and extent of the Australia 2019–20 Eucalyptus forest fires are not the legacy of forest management. Nat. Ecol. Evol. 5, 1003–1010 (2021).Article 

Google Scholar 
Lindenmayer, D. B., Kooyman, R., Taylor, C., Ward, M. & Watson, J. Recent Australian wildfires made worse by logging and associated forest management. Nat. Ecol. Evol. 4, 898–900 (2020).Article 

Google Scholar 
Gould, J. S., Knight, I. & Sullivan, A. L. Physical modelling of leaf scorch height from prescribed fires in young Eucalyptus sieberi regrowth forests in South-Eastern Australia. Int. J. Wildl. Fire 7, 7–20 (1997).Article 

Google Scholar 
Keith, D. Ocean Shores to Desert Dunes: The Native Vegetation of NSW and the ACT (Department of Environment and Conservation NSW, 2004).Burrows, N. Predicting canopy scorch height in jarrah forests. CALM Sci. 2, 267–274 (1997).
Google Scholar 
Penney, G., Habibi, D. & Cattani, M. Firefighter tenability and its influence on wildfire suppression. Fire Saf. J. 106, 38–51 (2019).Article 

Google Scholar 
Sharples, J. J. et al. Natural hazards in Australia: extreme bushfire. Clim. Change 139, 85–99 (2016).Article 

Google Scholar 
Attiwill, P. M. et al. Timber harvesting does not increase fire risk and severity in wet eucalypt forests of Southern Australia. Conserv. Lett. 7, 341–354 (2014).Article 

Google Scholar 
Lindenmayer, D., Taylor, C. & Blanchard, W. Empirical analyses of the factors influencing fire severity in southeastern Australia. Ecosphere 12, e03721 (2021).Article 

Google Scholar 
Taylor, C., Blanchard, W. & Lindenmayer, D. B. Does forest thinning reduce fire severity in Australian eucalypt forests? Conserv. Lett. 14, e12766 (2020).
Google Scholar 
Taylor, C., McCarthy, M. A. & Lindenmayer, D. B. Non-linear effects of stand age on fire severity. Conserv. Lett. 7, 355–370 (2014).Article 

Google Scholar 
Furlaud, J. M., Prior, L. D., Williamson, G. J. & Bowman, D. M. J. S. Fire risk and severity decline with stand development in Tasmanian giant Eucalyptus forest. For. Ecol. Manag. 502, 119724 (2021).Article 

Google Scholar 
Price, O. F. & Bradstock, R. A. The efficacy of fuel treatment in mitigating property loss during wildfires: insights from analysis of the severity of the catastrophic fires in 2009 in Victoria, Australia. J. Environ. Manag. 113, 146–157 (2012).Article 

Google Scholar 
Taylor, C. & Lindenmayer, D. B. The adequacy of Victoria’s protected areas for conserving its forest-dependent fauna. Austral Ecol. 44, 1076–1090 (2019).Article 

Google Scholar 
Taylor, C., Blanchard, W. & Lindenmayer, D. B. What are the relationships between thinning and fire severity? Austral Ecol. https://doi.org/10.1111/aec.13096 (2021).La Sala, A. Thinning Regrowth Eucalypts Native Forest Silviculture Technical Bulletin No. 13 (Forestry Tasmania, 2001).Cary, G. J., Blanchard, W., Foster, C. N. & Lindenmayer, D. B. Effects of altered fire intervals on critical timber production and conservation values. Int. J. Wildl. Fire 30, 322–328 (2021).Article 

Google Scholar 
Filkov, A. I. et al. The determinants of crown fire runs during extreme wildfires in broadleaf forests in Australia. Adv. For. Fire Res. https://doi.org/10.14195/978-989-26-16-506_190; http://hdl.handle.net/10316.2/44517 (2018).Lindenmayer, D. B., Hobbs, R. J., Likens, G. E., Krebs, C. J. & Banks, S. C. Newly discovered landscape traps produce regime shifts in wet forests. Proc. Natl Acad. Sci. USA 108, 15887–15891 (2011).CAS 
Article 

Google Scholar  More

Arneth, A. et al. Terrestrial biogeochemical feedbacks in the climate system. Nat. Geosci. 3, 525–532 (2010).CAS 
Article 

Google Scholar 
Green, J. K. et al. Regionally strong feedbacks between the atmosphere and terrestrial biosphere. Nat. Geosci. 10, 410–414 (2017).CAS 
Article 

Google Scholar 
Heimann, M. & Reichstein, M. Terrestrial ecosystem carbon dynamics and climate feedbacks. Nature 451, 289–292 (2008).CAS 
Article 

Google Scholar 
Beer, C. et al. Temporal and among-site variability of inherent water use efficiency at the ecosystem level. Glob. Biogeochem. Cycles 23, 1–13 (2009).Article 

Google Scholar 
Keenan, T. F. et al. Increase in forest water-use efficiency as atmospheric carbon dioxide concentrations rise. Nature 499, 324–327 (2013).CAS 
Article 

Google Scholar 
Frank, D. C. et al. Water-use efficiency & transpiration across European forests during the Anthropocene. Nat. Clim. Change 5, 579–583 (2015).CAS 
Article 

Google Scholar 
Mastrotheodoros, T. et al. Linking plant functional trait plasticity and the large increase in forest water use efficiency. J. Geophys. Res. Biogeosci. 122, 2393–2408 (2017).Article 

Google Scholar 
Lavergne, A. et al. Observed and modelled historical trends in the water-use efficiency of plants and ecosystems. Glob. Change Biol. 25, 2242–2257 (2019).Article 

Google Scholar 
Huxman, T. E. et al. Convergence across biomes to a common rain-use efficiency. Nature 429, 651–654 (2004).CAS 
Article 

Google Scholar 
Yang, Y. et al. Contrasting responses of water use efficiency to drought across global terrestrial ecosystems. Sci. Rep. 6, 23284 (2016).CAS 
Article 

Google Scholar 
Huang, L. et al. A global examination of the response of ecosystem water-use efficiency to drought based on MODIS data. Sci. Total Environ. 601–602, 1097–1107 (2017).Article 

Google Scholar 
Reichstein, M. et al. Severe drought effects on ecosystem CO2 and H2O fluxes at three Mediterranean evergreen sites: revision of current hypotheses? Glob. Change Biol. 8, 999–1017 (2002).Article 

Google Scholar 
Reichstein, M. et al. Inverse modeling of seasonal drought effects on canopy CO2/H2O exchange in three Mediterranean ecosystems. J. Geophys. Res. Atmos. 108, 4726 (2003).Article 

Google Scholar 
Cooley, S. S. et al. Assessing regional drought impacts on vegetation and evapotranspiration: a case study in Guanacaste, Costa Rica. Ecol. Appl. 29, e01834 (2019).Article 

Google Scholar 
Medrano, H., Flexas, J. & Galmés, J. Variability in water use efficiency at the leaf level among Mediterranean plants with different growth forms. Plant Soil 317, 17–29 (2008).Article 

Google Scholar 
Soh, W. K. et al. Rising CO2 drives divergence in water use efficiency of evergreen and deciduous plants. Sci. Adv. 5, eaax7906 (2019).CAS 
Article 

Google Scholar 
Wang, M., Chen, Y., Wu, X. & Bai, Y. Forest-type-dependent water use efficiency trends across the northern hemisphere. Geophys. Res. Lett. 45, 8283–8293 (2018).Article 

Google Scholar 
Enquist, B. et al. Scaling from traits to ecosystems: developing a general trait driver theory via integrating trait-based and metabolic scaling theories. Adv. Ecol. Res. 52, 249–318 (2015).Article 

Google Scholar 
Gross, N. et al. Functional trait diversity maximizes ecosystem multifunctionality. Nat. Ecol. Evol. 1, 0132 (2017).Article 

Google Scholar 
Bagousse‐Pinguet, Y. L. et al. Testing the environmental filtering concept in global drylands. J. Ecol. 105, 1058–1069 (2017).Article 

Google Scholar 
Ponce Campos, G. E. et al. Ecosystem resilience despite large-scale altered hydroclimatic conditions. Nature 494, 349–352 (2013).CAS 
Article 

Google Scholar 
Fisher, J. B. et al. The future of evapotranspiration: global requirements for ecosystem functioning, carbon and climate feedbacks, agricultural management, and water resources. Water Resour. Res. 53, 2618–2626 (2017).Article 

Google Scholar 
Xue, B.-L. et al. Global patterns, trends, and drivers of water use efficiency from 2000 to 2013. Ecosphere 6, art174 (2015).Article 

Google Scholar 
Fisher, J. B. et al. ECOSTRESS: NASA’s next generation mission to measure evapotranspiration from the International Space Station. Water Resour. Res. 56, e2019WR026058 (2020).Article 

Google Scholar 
Higgins, M. A. et al. Geological control of floristic composition in Amazonian forests. J. Biogeogr. 38, 2136–2149 (2011).Article 

Google Scholar 
De Kauwe, M. G., Keenan, T. F., Medlyn, B. E., Prentice, I. C. & Terrer, C. Satellite based estimates underestimate the effect of CO2 fertilization on net primary productivity. Nat. Clim. Change 6, 892–893 (2016).Article 

Google Scholar 
Huang, M. et al. Seasonal responses of terrestrial ecosystem water-use efficiency to climate change. Glob. Change Biol. 22, 2165–2177 (2016).Article 

Google Scholar 
Lin, Y.-S. et al. Optimal stomatal behaviour around the world. Nat. Clim. Change 5, 459–464 (2015).CAS 
Article 

Google Scholar 
Medlyn, B. E. et al. Reconciling the optimal and empirical approaches to modelling stomatal conductance. Glob. Change Biol. 17, 2134–2144 (2011).Article 

Google Scholar 
Peters, W. et al. Increased water-use efficiency and reduced CO2 uptake by plants during droughts at a continental scale. Nat. Geosci. 11, 744–748 (2018).CAS 
Article 

Google Scholar 
Cheng, L. et al. Recent increases in terrestrial carbon uptake at little cost to the water cycle. Nat. Commun. 8, 110 (2017).Article 

Google Scholar 
Fisher, J. B. ECOsystem Spaceborne Thermal Radiometer Experiment on Space Station (ECOSTRESS): Level-3 Evapotranspiration L3(ET_PT-JPL) Algorithm Theoretical Basis Document. Jet Propulsion Laboratory, California Institute of Technology (2018).Running, S. W. et al. A continuous satellite-derived measure of global terrestrial primary production. BioScience 54, 547–560 (2004).Article 

Google Scholar 
Heinsch, F. et al. Evaluation of remote sensing based terrestrial productivity from MODIS using regional tower eddy flux network observations. IEEE Trans. Geosci. Remote Sens. 44, 1908–1925 (2006).Article 

Google Scholar 
Zhao, M., Heinsch, F., Nemani, R. & Running, S. Improvements of the MODIS terrestrial gross and net primary production global data set. Remote Sens. Environ. 95, 164–176 (2005).Article 

Google Scholar 
Ryu, Y. et al. Integration of MODIS land and atmosphere products with a coupled-process model to estimate gross primary productivity and evapotranspiration from 1 km to global scales. Glob. Biogeochem. Cycles 25, GB4017 (2011).Article 

Google Scholar  More

Xu, Y. et al. Annual oil palm plantation maps in Malaysia and Indonesia from 2001 to 2016. Earth Syst. Sci. Data 12, 847–867 (2020).Article 

Google Scholar 
Meijaard, E. et al. The environmental impacts of palm oil in context. Nat. Plants 6, 1418–1426 (2020).Article 

Google Scholar 
Guillaume, T. et al. Carbon costs and benefits of Indonesian rainforest conversion to plantations. Nat. Commun. 9, 2388 (2018).Article 

Google Scholar 
Ordway, E. M. & Asner, G. P. Carbon declines along tropical forest edges correspond to heterogeneous effects on canopy structure and function. Proc. Natl Acad. Sci. USA 117, 7863–7870 (2020).CAS 
Article 

Google Scholar 
Hansen, M. C. et al. High-resolution global maps of 21st-century forest cover change. Science 342, 850 (2013).CAS 
Article 

Google Scholar 
Santoro, M. et al. The global forest above-ground biomass pool for 2010 estimated from high-resolution satellite observations. Earth Syst. Sci. Data 13, 3927–3950 (2021).Article 

Google Scholar 
The World Database on Protected Areas (WDPA) (UNEP-WCMC and IUCN, accessed 12 February 2020); www.protectedplanet.netMahmud, A., Rehrig, M. & Hills, G. Improving the Livelihoods of Palm Oil Smallholders: The Role of the Private Sector (FSG, 2010).Lasco, R. Forest carbon budgets in Southeast Asia following harvesting and land cover change. Sci. China 45, 55–64 (2002).Article 

Google Scholar 
Historical Greenhouse Gas Emissions (Climate Watch, accessed 6 October 2021); https://www.climatewatchdata.org/Euler, M., Schwarze, S., Siregar, H. & Qaim, M. Oil palm expansion among smallholder farmers in Sumatra, Indonesia. J. Agric. Econ. 67, 658–676 (2016).Article 

Google Scholar 
Donofrio, S., Rothrock, P. & Leonard, J. J. F. T. Supply Change: Tracking Corporate Commitments to Deforestation-free SupplyChains, 2017 (Forest Trends, 2017).Rist, L., Feintrenie, L. & Levang, P. The livelihood impacts of oil palm: smallholders in Indonesia. Biodivers. Conserv. 19, 1009–1024 (2010).Article 

Google Scholar 
Saadun, N. et al. Socio-ecological perspectives of engaging smallholders in environmental-friendly palm oil certification schemes. Land Use Policy 72, 333–340 (2018).Article 

Google Scholar 
Hansen, M. C., Stehman, S. V. & Potapov, P. V. Quantification of global gross forest cover loss. Proc. Natl Acad. Sci. USA 107, 8650 (2010).CAS 
Article 

Google Scholar 
Santoro, M. & Cartus, O. ESA Biomass Climate Change Initiative (Biomass_cci): Global datasets of forest above-ground biomass for the year 2017 v.1 (Centre for Environmental Data Analysis, 2019); https://doi.org/10.5285/bedc59f37c9545c981a839eb552e4084Busch, J. et al. Reductions in emissions from deforestation from Indonesia’s moratorium on new oil palm, timber, and logging concessions. Proc. Natl Acad. Sci. USA 112, 1328–1333 (2015).CAS 
Article 

Google Scholar 
McGarigal, K., Cushman, S. A. & Ene, E. FRAGSTATS v.4: spatial pattern analysis program for categorical and continuous maps (Univ. Massachusetts, 2012). More

Brown MV, Philip GK, Bunge JA, Smith MC, Bissett A, Lauro FM, et al. Microbial community structure in the North Pacific ocean. ISME J. 2009;3:1374–86.CAS 
Article 

Google Scholar 
Chénard C, Wijaya W, Vaulot D, dos Santos AL, Martin P, Kaur A, et al. Temporal and spatial dynamics of Bacteria, Archaea and protists in equatorial coastal waters. Sci Rep. 2019;9:1–13.Article 

Google Scholar 
Yeh YC, McNichol J, Needham DM, Fichot EB, Berdjeb L, Fuhrman JA. Comprehensive single‐PCR 16S and 18S rRNA community analysis validated with mock communities, and estimation of sequencing bias against 18S. Environ Microbiol. 2021;23:2340–3250.Article 

Google Scholar 
Needham DM, Fuhrman JA. Pronounced daily succession of phytoplankton, archaea and bacteria following a spring bloom. Nat. Microbiol. 2016;1:16005.CAS 
Article 

Google Scholar 
Parada AE, Needham DM, Fuhrman JA. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol. 2016;18:1403–14.CAS 
Article 

Google Scholar 
Needham DM, Fichot EB, Wang E, Berdjeb L, Cram JA, Fichot CG, et al. Dynamics and interactions of highly resolved marine plankton via automated high-frequency sampling. ISME J. 2018;12:2417.CAS 
Article 

Google Scholar 
Steinberg DK, Carlson CA, Bates NR, Johnson RJ, Michaels AF, Knap AH. Overview of the US JGOFS Bermuda Atlantic Time-series Study (BATS): a decade-scale look at ocean biology and biogeochemistry. Deep Sea Res Part II Top Stud Oceanogr. 2001;48:1405–47.CAS 
Article 

Google Scholar 
Karl DM, Church MJ. Microbial oceanography and the Hawaii Ocean Time-series programme. Nat Rev Microbiol. 2014;12:699–713.CAS 
Article 

Google Scholar 
Mestre M, Höfer J, Sala MM, Gasol JM. Seasonal variation of bacterial diversity along the marine particulate matter continuum. Front Microbiol. 2020;11:1590.Article 

Google Scholar 
Cram JA, Chow C-ET, Sachdeva R, Needham DM, Parada AE, Steele JA, et al. Seasonal and interannual variability of the marine bacterioplankton community throughout the water column over ten years. ISME J. 2015;9:563–80.Article 

Google Scholar 
Berelson WM. The flushing of two deep‐sea basins, southern California borderland. Limnol Oceanogr. 1991;36:1150–66.CAS 
Article 

Google Scholar 
Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–3.CAS 
Article 

Google Scholar 
Traving SJ, Kellogg CT, Ross T, McLaughlin R, Kieft B, Ho GY, et al. Prokaryotic responses to a warm temperature anomaly in northeast subarctic Pacific waters. Commun Biology. 2021;4:1–12.Article 

Google Scholar 
Butler TM, Wilhelm A-C, Dwyer AC, Webb PN, Baldwin AL, Techtmann SM. Microbial community dynamics during lake ice freezing. Scient Rep. 2019;9:1–11.
Google Scholar 
LeBrun ES, King RS, Back JA, Kang S. Microbial community structure and function decoupling across a phosphorus gradient in streams. Microb Ecol. 2018;75:64–73.CAS 
Article 

Google Scholar 
McNichol J, Berube PM, Biller SJ, Fuhrman JA. Evaluating and improving small subunit rRNA PCR primer coverage for bacteria, archaea, and eukaryotes using metagenomes from global ocean surveys. Msystems. 2021;6:e00565–21.CAS 
Article 

Google Scholar 
De Bie T, De Meester L, Brendonck L, Martens K, Goddeeris B, Ercken D, et al. Body size and dispersal mode as key traits determining metacommunity structure of aquatic organisms. Ecol Lett. 2012;15:740–7.Article 

Google Scholar 
Soininen J, Korhonen JJ, Karhu J, Vetterli A. Disentangling the spatial patterns in community composition of prokaryotic and eukaryotic lake plankton. Limnol. Oceanogr. 2011;56:508–20.Article 

Google Scholar 
Wu W, Lu H-P, Sastri A, Yeh Y-C, Gong G-C, Chou W-C, et al. Contrasting the relative importance of species sorting and dispersal limitation in shaping marine bacterial versus protist communities. ISME J. 2018;12:485–94.Article 

Google Scholar 
Kraemer S, Ramachandran A, Colatriano D, Lovejoy C, Walsh DA. Diversity and biogeography of SAR11 bacteria from the Arctic Ocean. ISME J. 2020;14:79–90.Article 

Google Scholar 
Tsementzi D, Wu J, Deutsch S, Nath S, Rodriguez-R LM, Burns AS, et al. SAR11 bacteria linked to ocean anoxia and nitrogen loss. Nature. 2016;536:179–83.CAS 
Article 

Google Scholar 
Brown MV, Lauro FM, DeMaere MZ, Muir L, Wilkins D, Thomas T, et al. Global biogeography of SAR11 marine bacteria. Mol Syst Biol. 2012;8:595.Article 

Google Scholar 
Giovannoni SJ. SAR11 bacteria: the most abundant plankton in the oceans. Ann Rev Mar Sci. 2017;9:231–55.Article 

Google Scholar 
Thrash JC, Temperton B, Swan BK, Landry ZC, Woyke T, DeLong EF, et al. Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype. ISME J. 2014;8:1440–51.Article 

Google Scholar 
Fernández-Gomez B, Richter M, Schüler M, Pinhassi J, Acinas SG, González JM, et al. Ecology of marine Bacteroidetes: a comparative genomics approach. ISME J. 2013;7:1026–37.Article 

Google Scholar 
Countway PD, Vigil PD, Schnetzer A, Moorthi SD, Caron DA. Seasonal analysis of protistan community structure and diversity at the USC Microbial Observatory (San Pedro Channel, North Pacific Ocean). Limnol Oceanogr. 2010;55:2381–96.Article 

Google Scholar 
Kim DY, Countway PD, Jones AC, Schnetzer A, Yamashita W, Tung C, et al. Monthly to interannual variability of microbial eukaryote assemblages at four depths in the eastern North Pacific. ISME J. 2014;8:515–30.Article 

Google Scholar 
Parris DJ, Ganesh S, Edgcomb VP, DeLong EF, Stewart FJ. Microbial eukaryote diversity in the marine oxygen minimum zone off northern Chile. Front Microbiol. 2014;5:543.Article 

Google Scholar 
Orsi W, Song YC, Hallam S, Edgcomb V. Effect of oxygen minimum zone formation on communities of marine protists. ISME J. 2012;6:1586–601.CAS 
Article 

Google Scholar 
Leibold MA, Holyoak M, Mouquet N, Amarasekare P, Chase JM, Hoopes MF, et al. The metacommunity concept: a framework for multi‐scale community ecology. Ecol Lett. 2004;7:601–13.Article 

Google Scholar 
Fuhrman JA, Hewson I, Schwalbach MS, Steele JA, Brown MV, Naeem S. Annually reoccurring bacterial communities are predictable from ocean conditions. Proc Natl Acad Sci USA. 2006;103:13104–9.CAS 
Article 

Google Scholar 
Chow C-ET, Sachdeva R, Cram JA, Steele JA, Needham DM, Patel A, et al. Temporal variability and coherence of euphotic zone bacterial communities over a decade in the Southern California Bight. ISME J. 2013;7:2259–73.CAS 
Article 

Google Scholar 
Parada AE, Fuhrman JA. Marine archaeal dynamics and interactions with the microbial community over 5 years from surface to seafloor. ISME J. 2017;11:2510–25.Article 

Google Scholar 
Mestre M, Ruiz-González C, Logares R, Duarte CM, Gasol JM, Sala MM. Sinking particles promote vertical connectivity in the ocean microbiome. Proc Natl Acad Sci USA. 2018;115:E6799–E807.CAS 
Article 

Google Scholar 
Mestre M, Ferrera I, Borrull E, Ortega‐Retuerta E, Mbedi S, Grossart HP, et al. Spatial variability of marine bacterial and archaeal communities along the particulate matter continuum. Mol Ecol. 2017;26:6827–40.CAS 
Article 

Google Scholar 
Wilson B, Müller O, Nordmann E-L, Seuthe L, Bratbak G, Øvreås L. Changes in marine prokaryote composition with season and depth over an Arctic polar year. Front Mar Sci. 2017;4:95.
Google Scholar 
Treusch AH, Vergin KL, Finlay LA, Donatz MG, Burton RM, Carlson CA, et al. Seasonality and vertical structure of microbial communities in an ocean gyre. ISME J. 2009;3:1148–63.Article 

Google Scholar 
Zinger L, Amaral-Zettler LA, Fuhrman JA, Horner-Devine MC, Huse SM, Welch DBM, et al. Global patterns of bacterial beta-diversity in seafloor and seawater ecosystems. PLoS ONE. 2011;6:e24570.CAS 
Article 

Google Scholar 
DeLong EF, Preston CM, Mincer T, Rich V, Hallam SJ, Frigaard N-U, et al. Community genomics among stratified microbial assemblages in the ocean’s interior. Science. 2006;311:496–503.CAS 
Article 

Google Scholar 
Agogué H, Lamy D, Neal PR, Sogin ML, Herndl GJ. Water mass‐specificity of bacterial communities in the North Atlantic revealed by massively parallel sequencing. Mol. Ecol. 2011;20:258–74.Article 

Google Scholar 
Walsh EA, Kirkpatrick JB, Rutherford SD, Smith DC, Sogin M, D’Hondt S. Bacterial diversity and community composition from seasurface to subseafloor. ISME J. 2016;10:979–89.Article 

Google Scholar 
Reji L, Tolar BB, Chavez FP, Francis CA. Depth-differentiation and seasonality of planktonic microbial assemblages in the Monterey Bay upwelling system. Front Microbiol. 2020;11:1075.Article 

Google Scholar 
Milici M, Vital M, Tomasch J, Badewien TH, Giebel HA, Plumeier I, et al. Diversity and community composition of particle‐associated and free‐living bacteria in mesopelagic and bathypelagic Southern Ocean water masses: Evidence of dispersal limitation in the Bransfield Strait. Limnol Oceanogr. 2017;62:1080–95.Article 

Google Scholar 
Crespo BG, Pommier T, Fernández‐Gómez B, Pedrós‐Alió C. Taxonomic composition of the particle‐attached and free‐living bacterial assemblages in the Northwest Mediterranean Sea analyzed by pyrosequencing of the 16S rRNA. Microbiologyopen. 2013;2:541–52.CAS 
Article 

Google Scholar 
Ganesh S, Parris DJ, DeLong EF, Stewart FJ. Metagenomic analysis of size-fractionated picoplankton in a marine oxygen minimum zone. ISME J. 2014;8:187–211.CAS 
Article 

Google Scholar 
Ghiglione J-F, Galand PE, Pommier T, Pedrós-Alió C, Maas EW, Bakker K, et al. Pole-to-pole biogeography of surface and deep marine bacterial communities. Proc Natl Acad Sci USA. 2012;109:17633–8.CAS 
Article 

Google Scholar 
Murillo AA, Ramírez-Flandes S, DeLong EF, Ulloa O. Enhanced metabolic versatility of planktonic sulfur-oxidizing γ-proteobacteria in an oxygen-deficient coastal ecosystem. Front Mar Sci. 2014;1:18.Article 

Google Scholar 
Hawley AK, Nobu MK, Wright JJ, Durno WE, Morgan-Lang C, Sage B, et al. Diverse Marinimicrobia bacteria may mediate coupled biogeochemical cycles along eco-thermodynamic gradients. Nat Commun. 2017;8:1–10.CAS 
Article 

Google Scholar 
Santoro AE, Buchwald C, McIlvin MR, Casciotti KL. Isotopic signature of N2O produced by marine ammonia-oxidizing archaea. Science. 2011;333:1282–5.CAS 
Article 

Google Scholar 
Aldunate M, De la Iglesia R, Bertagnolli AD, Ulloa O. Oxygen modulates bacterial community composition in the coastal upwelling waters off central Chile. Deep Sea Res Part II Top Stud Oceanogr. 2018;156:68–79.CAS 
Article 

Google Scholar 
Duret MT, Lampitt RS, Lam P. Prokaryotic niche partitioning between suspended and sinking marine particles. Environ Microbiol Rep. 2019;11:386–400.CAS 
Article 

Google Scholar 
Lindh MV, Sjöstedt J, Andersson AF, Baltar F, Hugerth LW, Lundin D, et al. Disentangling seasonal bacterioplankton population dynamics by high‐frequency sampling. Environ Microbiol. 2015;17:2459–76.Article 

Google Scholar 
Teeling H, Fuchs BM, Bennke CM, Krueger K, Chafee M, Kappelmann L, et al. Recurring patterns in bacterioplankton dynamics during coastal spring algae blooms. elife. 2016;5:e11888.Article 

Google Scholar 
Buchan A, LeCleir GR, Gulvik CA, González JM. Master recyclers: features and functions of bacteria associated with phytoplankton blooms. Nat Rev Microbiol. 2014;12:686–98.CAS 
Article 

Google Scholar 
Giovannoni SJ, Tripp HJ, Givan S, Podar M, Vergin KL, Baptista D, et al. Genome streamlining in a cosmopolitan oceanic bacterium. Science. 2005;309:1242–5.CAS 
Article 

Google Scholar 
Cram JA, Xia LC, Needham DM, Sachdeva R, Sun F, Fuhrman JA. Cross-depth analysis of marine bacterial networks suggests downward propagation of temporal changes. ISME J. 2015;9:2573–86.Article 

Google Scholar 
Salazar G, Cornejo‐Castillo FM, Borrull E, Díez‐Vives C, Lara E, Vaqué D, et al. Particle‐association lifestyle is a phylogenetically conserved trait in bathypelagic prokaryotes. Mol Ecol. 2015;24:5692–706.Article 

Google Scholar 
Mohit V, Archambault P, Toupoint N, Lovejoy C. Phylogenetic differences in attached and free-living bacterial communities in a temperate coastal lagoon during summer, revealed via high-throughput 16S rRNA gene sequencing. Appl Environ Microbiol. 2014;80:2071–83.Article 

Google Scholar 
Rieck A, Herlemann DP, Jürgens K, Grossart H-P. Particle-associated differ from free-living bacteria in surface waters of the Baltic Sea. Front Microbiol. 2015;6:1297.Article 

Google Scholar 
Pachiadaki MG, Brown JM, Brown J, Bezuidt O, Berube PM, Biller SJ, et al. Charting the complexity of the marine microbiome through single-cell genomics. Cell. 2019;179:1623–35. e11.CAS 
Article 

Google Scholar 
Giner CR, Pernice MC, Balagué V, Duarte CM, Gasol JM, Logares R, et al. Marked changes in diversity and relative activity of picoeukaryotes with depth in the world ocean. ISME J. 2020;14:437–49.Article 

Google Scholar 
Countway PD, Gast RJ, Dennett MR, Savai P, Rose JM, Caron DA. Distinct protistan assemblages characterize the euphotic zone and deep sea (2500 m) of the western North Atlantic (Sargasso Sea and Gulf Stream). Environ Microbiol. 2007;9:1219–32.CAS 
Article 

Google Scholar 
Ollison GA, Hu SK, Mesrop LY, DeLong EF, Caron DA. Come rain or shine: depth not season shapes the active protistan community at station ALOHA in the North Pacific Subtropical Gyre. Deep Sea Res Part I Oceanogr Res Pap. 2021;170:103494.Article 

Google Scholar 
Schnetzer A, Moorthi SD, Countway PD, Gast RJ, Gilg IC, Caron DA. Depth matters: microbial eukaryote diversity and community structure in the eastern North Pacific revealed through environmental gene libraries. Deep Sea Res Part I Oceanogr Res Pap. 2011;58:16–26.Article 

Google Scholar 
Martin P, Allen JT, Cooper MJ, Johns DG, Lampitt RS, Sanders R, et al. Sedimentation of acantharian cysts in the Iceland Basin: strontium as a ballast for deep ocean particle flux, and implications for acantharian reproductive strategies. Limnol Oceanogr. 2010;55:604–14.CAS 
Article 

Google Scholar 
Lampitt R, Salter I, Johns D. Radiolaria: Major exporters of organic carbon to the deep ocean. Glob Biogeochem Cycle. 2009;23:GB1010.Article 

Google Scholar 
Skovgaard A, Massana R, Balague V, Saiz E. Phylogenetic position of the copepod-infesting parasite Syndinium turbo (Dinoflagellata, Syndinea). Protist. 2005;156:413–23.CAS 
Article 

Google Scholar 
Bachvaroff TR, Kim S, Guillou L, Delwiche CF, Coats DW. Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis. Appl Environ Microbiol. 2012;78:334–45.CAS 
Article 

Google Scholar 
Guillou L, Viprey M, Chambouvet A, Welsh R, Kirkham A, Massana R, et al. Widespread occurrence and genetic diversity of marine parasitoids belonging to Syndiniales (Alveolata). Environ Microbiol. 2008;10:3349–65.CAS 
Article 

Google Scholar 
Berdjeb L, Parada A, Needham DM, Fuhrman JA. Short-term dynamics and interactions of marine protist communities during the spring–summer transition. ISME J. 2018;12:1907.Article 

Google Scholar 
Hu SK, Connell PE, Mesrop LY, Caron DA. A hard day’s night: diel shifts in microbial eukaryotic activity in the north pacific subtropical gyre. Front Mar Sci. 2018;5:351.Article 

Google Scholar 
Clarke LJ, Bestley S, Bissett A, Deagle BE. A globally distributed Syndiniales parasite dominates the Southern Ocean micro-eukaryote community near the sea-ice edge. ISME J. 2019;13:734–7.CAS 
Article 

Google Scholar 
De Vargas C, Audic S, Henry N, Decelle J, Mahé F, Logares R, et al. Eukaryotic plankton diversity in the sunlit ocean. Science. 2015;348:1261605.Article 

Google Scholar 
Pernice MC, Giner CR, Logares R, Perera-Bel J, Acinas SG, Duarte CM, et al. Large variability of bathypelagic microbial eukaryotic communities across the world’s oceans. ISME J. 2016;10:945–58.Article 

Google Scholar 
Crutsinger GM, Collins MD, Fordyce JA, Gompert Z, Nice CC, Sanders NJ. Plant genotypic diversity predicts community structure and governs an ecosystem process. Science. 2006;313:966–8.CAS 
Article 

Google Scholar 
Hawkins BA, Porter EE. Does herbivore diversity depend on plant diversity? The case of California butterflies. Am Nat. 2003;161:40–9.Article 

Google Scholar 
Scherber C, Eisenhauer N, Weisser WW, Schmid B, Voigt W, Fischer M, et al. Bottom-up effects of plant diversity on multitrophic interactions in a biodiversity experiment. Nature. 2010;468:553–6.CAS 
Article 

Google Scholar 
Yang JW, Wu W, Chung C-C, Chiang K-P, Gong G-C, Hsieh C-h Predator and prey biodiversity relationship and its consequences on marine ecosystem functioning—interplay between nanoflagellates and bacterioplankton. ISME J. 2018;12:1532–42.Article 

Google Scholar 
Fuhrman JA, Comeau DE, Hagström Å, Chan AM. Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies. Appl Environ Microbiol. 1988;54:1426–9.CAS 
Article 

Google Scholar 
Lie AA, Kim DY, Schnetzer A, Caron DA. Small-scale temporal and spatial variations in protistan community composition at the San Pedro Ocean Time-series station off the coast of southern California. Aquat Microb Ecol. 2013;70:93–110.Article 

Google Scholar 
Yeh Y-C, Needham DM, Sieradzki ET, Fuhrman JA. Taxon disappearance from microbiome analysis reinforces the value of mock communities as a standard in every sequencing run. MSystems. 2018;3:e00023–18.Article 

Google Scholar 
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl Acids Res. 2013;41:D590–D6.CAS 
Article 

Google Scholar 
Guillou L, Bachar D, Audic S, Bass D, Berney C, Bittner L, et al. The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucl Acids Res. 2013;41:D579–D604.
Google Scholar 
Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37:852–7.CAS 
Article 

Google Scholar 
Decelle J, Romac S, Stern RF, Bendif EM, Zingone A, Audic S, et al. Phyto REF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy. Mol Ecol Resour. 2015;15:1435–45.CAS 
Article 

Google Scholar 
Oksanen J, Blanchet F, Friendly M, Kindt R, Legendre P, McGlinn D, et al. Vegan: community ecology package. R package version 2.5-7. 2020. https://CRAN.R-project.org/package=vegan.Wickham H. ggplot2-elegant graphics for data analysis. Cham, Switzerland: Springer International Publishing; 2016.Kolde R. Pheatmap: pretty heatmaps. R Package Version. 2012;1:726.
Google Scholar 
Schloerke B, Crowley J, Cook D. Package ‘GGally’. Extension to ‘ggplot2’See. 2018;713. More