Received: 9 November 2020 | Accepted: 25 May 2021 DOI: 10.1111/gcb.15752 PRIMARY RESEARCH ARTICLE A “Dirty” Footprint: Macroinvertebrate diversity in Amazonian Anthropic Soils Wilian C. Demetrio1 | Ana C. Conrado2 | Agno N. S. Acioli3 | Alexandre C. Ferreira4 | Marie L. C. Bartz5 | Samuel W. James6 | Elodie da Silva7 | Lilianne S. Maia1 | Gilvan C. Martins8 | Rodrigo S. Macedo9 | David W. G. Stanton10 | Patrick Lavelle11 | Elena Velasquez12 | Anne Zangerlé13 | Rafaella Barbosa14 | Sandra C. Tapia-Coral15 | Aleksander W. Muniz8 | Alessandra Santos1 | Talita Ferreira1 | Rodrigo F. Segalla1 | Thibaud Decaëns16 | Herlon S. Nadolny1 | Clara P. Peña-Venegas17 | Cláudia M. B. F. Maia7 | Amarildo Pasini18 | André F. Mota2 | Paulo S. Taube Júnior19 | Telma A. C. Silva20 | Lilian Rebellato19 | Raimundo C. de Oliveira Júnior21 | Eduardo G. Neves22 | Helena P. Lima23 | Rodrigo M. Feitosa4 | Pablo Vidal Torrado24 | Doyle McKey16 | Charles R. Clement20 | Myrtle P. Shock19 | Wenceslau G. Teixeira25 | Antônio C. V. Motta1 | Vander F. Melo1 | Jeferson Dieckow1 | Marilice C. Garrastazu7 | | Luís Cunha5,27 Leda S. Chubatsu2 | TPI Network* | Peter Kille26 | George G. Brown1,7 Department of Soil Science, Federal University of Paraná, Curitiba, PR, Brazil 1 2 Biochemistry Department, Federal University of Paraná, Curitiba, PR, Brazil 3 Federal University of Amazonas, Manaus, AM, Brazil 4 Entomology Department, Federal University of Paraná, Curitiba, PR, Brazil 5 Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal 6 Maharishi International University, Fairfield, IA, USA 7 Embrapa Florestas, Colombo, PR, Brazil 8 Embrapa Amazônia Ocidental, Manaus, AM, Brazil 9 Instituto Nacional do Semiárido, Campina Grande, PB, Brazil Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Stockholm, Sweden 10 Institut de Recherche pour le Développement, Cali, Colombia 11 12 Universidad Nacional de Colombia, Palmira, Colombia 13 Ministère de l’Agriculture, de la Viticulture et de la Protection des consommateurs, Luxembourg, Luxembourg 14 Centro Universitário do Norte, Manaus, AM, Brazil 15 Servicio Nacional de Aprendizaje, SENA Regional Amazonas, Leticia, Colombia 16 CEFE, Univ Montpellier, CNRS, EPHE, IRD, Univ Paul-Valéry Montpellier, Montpellier, France 17 Instituto Amazónico de Investigaciones Científicas SINCHI, Leticia, Colombia 18 19 Universidade Estadual de Londrina, Londrina, PR, Brazil Universidade Federal do Oeste do Pará, Pará, Brazil 20 Instituto Nacional de Pesquisas da Amazônia, Manaus, AM, Brazil *For a complete list of authors included in this network, please refer to the list of participants in the Web site http://tpinet.org. George G. Brown and Luís Cunha contributed equally to this work and should be considered as senior authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Global Change Biology published by John Wiley & Sons Ltd. Glob Change Biol. 2021;00:1–17. wileyonlinelibrary.com/journal/gcb | 1 2 | DEMETRIO ET al. Embrapa Amazônia Oriental, Santarém, PA, Brazil 21 22 Museu de Arqueologia e Etnologia, Universidade de São Paulo, São Paulo, SP, Brazil 23 Museu Paraense Emílio Goeldi, Belém, PA, Brazil 24 Soil Science Department, Escola Superior de Agricultura Luís de Queiroz, Universidade de São Paulo, Piracicaba, SP, Brazil 25 Embrapa Solos, Rio de Janeiro, RJ, Brazil 26 School of Biosciences, Cardiff University, Cardiff, CF, UK 27 School of Applied Sciences, University of South Wales, Pontypridd, CF, UK Correspondence George G. Brown, Embrapa Florestas, Colombo, PR, 83411- 000, Brazil. Email: george.brown@embrapa.br Luís Cunha, Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal. Email: luis.cunha@uc.pt Present address Wilian C. Demetrio, INPE – National Institute for Space Research, São José dos Campos, SP 12227- 010, Brazil Funding information Natural Environment Research Council, Grant/Award Number: NE/M017656/1; Newton Fund, Grant/Award Number: NE/N000323/1; European Union Horizon 2020 Marie-Curie, Grant/Award Number: MSCA-IF-2014- GF-660378 and 796877; CNPq, Grant/Award Number: 140260/2016-1, 302462/20163, 310690/2017- 0, 303477/2018- 0, 307179/2013-3, 307486/2013-3, 400533/2014-6, 401824/2013-6, 150748/2014- 0 and 165702/2015- 0; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Grant/Award Number: PVE A115/2013; Fundação Araucária, Grant/Award Number: 45166.460.32093.02022015; NAS/ USAID, Grant/Award Number: AID-OAAA-11- 0001 1 | Abstract Amazonian rainforests, once thought to be pristine wilderness, are increasingly known to have been widely inhabited, modified, and managed prior to European arrival, by human populations with diverse cultural backgrounds. Amazonian Dark Earths (ADEs) are fertile soils found throughout the Amazon Basin, created by pre-Columbian societies with sedentary habits. Much is known about the chemistry of these soils, yet their zoology has been neglected. Hence, we characterized soil fertility, macroinvertebrate communities, and their activity at nine archeological sites in three Amazonian regions in ADEs and adjacent reference soils under native forest (young and old) and agricultural systems. We found 673 morphospecies and, despite similar richness in ADEs (385 spp.) and reference soils (399 spp.), we identified a tenacious pre-Columbian footprint, with 49% of morphospecies found exclusively in ADEs. Termite and total macroinvertebrate abundance were higher in reference soils, while soil fertility and macroinvertebrate activity were higher in the ADEs, and associated with larger earthworm quantities and biomass. We show that ADE habitats have a unique pool of species, but that modern land use of ADEs decreases their populations, diversity, and contributions to soil functioning. These findings support the idea that humans created and sustained high-fertility ecosystems that persist today, altering biodiversity patterns in Amazonia. KEYWORDS Amazonian Dark Earths, ants, archeological sites, disturbance, earthworms, land-use change, soil fauna, soil fertility, termites, Terra Preta I NTRO D U C TI O N But humans have been modifying Amazonian biodiversity patterns over millennia. Native Amazonians created areas with high The Amazon basin still contains the largest continuous and rel- concentrations of useful trees and hyperdominance of some species, atively well-preserved tract of tropical forest on the planet. often associated with archeological sites (Levis et al., 2017, 2018; Ter However, deforestation rates have been increasing over the last Steege et al., 2013). Furthermore, occupations of some indigenous decade, resulting in the loss of an estimated 11.088 km2 of natural societies, beginning at least 6500 years ago, created fertile soils, lo- vegetation in 2020 alone (INPE, 2021). Many forested areas have cally called Amazonian Dark Earths (ADEs) or “Terra Preta de Índio” become highly fragmented and may be reaching tipping points in Portuguese (Clement et al., 2015; Glaser, 2007; Glaser & Birk, where biodiversity and ecosystem functions may be dramatically 2012; McMichael et al., 2014; Watling et al., 2018; Figure 1b) that affected (Barkhordarian et al., 2018; Decaëns et al., 2018), poten- may occupy from 0.1 (Sombroek et al., 2003) up to 3% (McMichael tially leading to cascading effects that impact ecosystem function- et al., 2014) of the surface area of Amazonia. They appear to be more ing over a much larger area (Lathuillière et al., 2018; Lawrence & common along major rivers (Figure 1a) but are also abundant in inter- Vandecar, 2015). fluvial areas (Clement et al., 2015; Levis et al., 2020). ADE sites tend | DEMETRIO ET al. 3 F I G U R E 1 Sampling strategy to assess soil fauna and soil fertility in Central (Iranduba), Southwestern (Porto Velho), and Lower (Belterra) Amazon. (a) Boundary of Amazon Basin (white line), showing municipalities where samples were taken (boundaries in yellow lines), and areas with large occurrence of Amazonian Dark Earths (ADEs, shaded in green), modified from Clement et al. (2015). Amazonia map background: Esri, DigitalGlobe, GeoEye, Earthstar Geographics, CNES/Airbus DS, USDA, USGS, AEX, Getmapping, Aerogrid, IGN, IGP, swisstopo, and the GIS User Community. (b) Soil profiles of analytically paired ADE and nearby reference (REF) soils. The direction of the arrow shows the increase in soil fertility; Photos G.C. Martins, R. Macedo. (c) Land-use systems sampled in each region, consisting in an intensification/ disturbance gradient including older secondary rainforest (>20 years, undisturbed), young regeneration forest (<20 years old), and recent agricultural systems (pasture, soybean, and maize). The direction of the arrow shows the increase in contemporary anthropogenic disturbance. Photos G.C. Martins, M. Bartz to have high contents of soil P, Ca, and pyrogenic-C (Glaser & Birk, habitat (explained by a pre-Columbian footprint) but also that (2) an- 2012; Lima et al., 2002; Sombroek et al., 2003), and host particular imal species richness, biomass, and activity, as well as nutrient con- communities of plants and soil microorganisms (Brossi et al., 2014; tents in these soils, would be determined by present-day land use. Taketani et al., 2013). However, up to now soil animal communities in these anthropic soils are practically unknown, having been the target of only three studies of limited geographic scope (all sites near Manaus), focusing on earthworms (Cunha et al., 2016) and soil arthropods (Sales et al., 2007; Soares et al., 2011). 2 | M ATE R I A L S A N D M E TH O DS 2.1 | Study sites Soil macroinvertebrates represent as much as 25% of overall known described species (Decaëns et al., 2006), and may easily sur- Our study was performed in three regions (central, lower, and pass 1 million species worldwide (Brown et al., 2018). However, soil southwestern Amazonia) of Brazilian Amazonia, with sampling con- animal communities have been little studied in megadiverse regions ducted in Iranduba county in central Amazonia, Belterra county such as the Amazonian rainforest (Barros et al., 2006; Franco et al., in lower Amazonia, and Porto Velho in southwestern Amazonia 2018; Marichal et al., 2014), and these habitats may be home to (Figure 1a; Table 1). All regions have a tropical monsoon (Köppen's thousands of described and still undescribed species (Brown et al., Am) or without dry season (Köppen's Af) climate, with a mean an- 2006), particularly smaller invertebrates such as nematodes and nual temperature of 24–26.7°C and precipitation between 2000 and mites (Franklin & Morais, 2006; Huang & Cares, 2006) but also mac- 2420 mm year−1 (Alvares et al., 2013). In each region, paired sites roinvertebrates (Mathieu, 2004). Furthermore, these invertebrates with ADEs and nearby non-anthropic REF soils (Figure 1b) were may be particularly susceptible to land-use changes such as defor- selected under different land-use systems (Figure 1c): native sec- estation (Decaëns et al., 2018; Franco et al., 2018; Mathieu et al., ondary vegetation (dense ombrophilous forest) classified as old sec- 2005) and can be used as bioindicators of both soil quality and of ondary forest when >20 years old, or young regeneration forest environmental disturbance (Gerlach et al., 2013; Lawton et al., 1998; when <20 years old, and agricultural systems of maize in Iranduba, Rousseau et al., 2013; Velásquez & Lavelle, 2019). soybean in Belterra, and introduced pasture in Porto Velho. The REF Hence, the aim of this study was to assess soil invertebrate mac- sites were located within a minimum distance of 150 m (soybean at rofauna communities and their activity in nine ADEs classified as Belterra) to a maximum distance of 1.3 km (pasture at Porto Velho) Anthrosols and nine non-anthropic reference Amazonian Acrisols, from the ADE sites, and maximum distance between the three sam- Ferralsols and Plinthosols (referred to in this paper as REF soils) pling locations within a region was 14 km (Embrapa sites to Tapajós under three land-use systems (LUS: old and young secondary forest National Forest sites in Belterra), totaling 18 sampled sites (3 re- and recent agricultural/pastoral systems; Figure 1c), to evaluate an- gions × 3 land-use systems × 2 soil types). thropic effects on soil biodiversity. We predicted that (1) soil biodi- One of the two old secondary forest sites in Belterra was at the versity and soil enrichment in anthropic soils would reflect a unique Embrapa Amazônia Oriental Belterra Experiment Station, whereas 4 | DEMETRIO ET al. TA B L E 1 Land-use system, age of modern human intervention, soil type, and soil category according to IUSS (2015) and location of the sites studied in three regions of Brazilian Amazonia Region State Land use Human interventiona Soil type Soil category Coordinates Iranduba AM Old forest >20 years old REF Xanthic Dystric Acrisol 3°14′49.00″S, 60°13′30.71″W Old forest >20 years old ADE Pretic Clayic Anthrosol 3°15′11.05″S, 60°13′45.03″W Young forest <20 years old REF Xanthic Dystric Acrisol 3°13′34.47″S, 60°16′23.60″W Young forest <20 years old ADE Pretic Clayic Anthrosol 3°13′49.23″S, 60°16′7.43″W Agricultural Current REF Xanthic Dystric Acrisol 3°13′31.31″S, 60°16′29.18″W Agricultural Current ADE Pretic Clayic Anthrosol 3°13′46.13″S, 60°16′7.32″W Old forest >20 years old REF Xanthic Dystric Ferralsol 2°47′4.59″S, 54°59′53.28″W Old forest >20 years old ADE Pretic Clayic Anthrosol 2°47′3.25″S, 54°59′59.77″W Old forest >20 years old REF Xanthic Dystric Acrisol 2°41′13.90″S, 54°55′3.30″W Old forest >20 years old ADE Pretic Clayic Anthrosol 2°41′7.18″S, 54°55′7.11″W Agricultural Current REF Xanthic Dystric Acrisol 2°41′3.56″S, 54°55′12.75″W Agricultural Current ADE Pretic Clayic Anthrosol 2°41′3.79″S, 54°55′7.90″W Young forest <20 years old REF Xanthic Dystric Plinthosol 8°52′11.50″S, 64°03′18.16″W Young forest <20 years old ADE Pretic Clayic Anthrosol 8°51′51.92″S, 64°03′48.03″W Young forest <20 years old REF Xanthic Dystric Ferralsol 8°50′49.52″S, 64°03′59.20″W Young forest <20 years old ADE Pretic Clayic Anthrosol 8°51′1.18″S, 64°04′3.07″W Agricultural Current REF Xanthic Dystric Ferralsol 8°52′35.30″S, 64°03′58.58″W Agricultural Current ADE Pretic Clayic Anthrosol 8°51′56.53″S, 64°03′40.67″W Belterra Porto Velho PA RO Abbreviations: ADE, Amazonian Dark Earth; REF, reference soil. a Age of modern human disturbance (land management). the other one was at the Tapajós National Forest, a site of previ- have begun ~1050–950 years bp (Macedo, 2014; Neves et al., 2004) ous work on ADEs (Maezumi et al., 2018). The old secondary for- and at Belterra ~530–450 years ests (ADE and REF) in Iranduba were at the Caldeirão Experimental Velho, ADE formation began much earlier (~6500 years Station of Embrapa Amazônia Ocidental and have been extensively et al., 2018). bp (Maezumi et al., 2018). At Porto bp; Watling studied in the past for soil fertility and pedogenesis (Alho et al., The agricultural fields with annual crops were under continuous 2019; Macedo et al., 2017), as well as for soil microbial diversity (at least 6 years) annual row cropping of maize (Iranduba) and soy- (Germano et al., 2012; Grossman et al., 2010; Lima et al., 2014, bean (Belterra) and had been planted <60 days prior to sampling, 2015; O’Neill et al., 2009; Taketani et al., 2013). Initial and partial using conventional tillage (Iranduba) or reduced tillage (Belterra). results of the earthworm data from the young and old forests, and The crops received the recommended doses of inorganic fertilizers the maize fields in Iranduba, were presented in an earlier publication and pesticides for each crop; all crops were planted using certified (Cunha et al., 2016). ADE formation in Iranduba was estimated to commercial seeds. The pastures at Porto Velho were around 9 year | DEMETRIO ET al. 5 old (REF) and 12 year old (ADE) and planted with Brachiaria (REF) individuals collected preserved in 96% ethanol. The extra termite and Paspalum (ADE) grasses. Soils at most REF sites were classified samples were collected in old secondary forests and young regen- according to the World Reference Base for Soil Resources—WRB eration forests (except at one of the REF young forests in Porto (IUSS, 2015) as dystrophic Ferralsols and Acrisols (Table 1), the two Velho), but not in the agricultural fields (maize, soybean, and pasture) most common soil types in Amazonia (Gardi et al., 2015). At one where there are very few termite colonies and the TSBF monoliths young regeneration forest site in Porto Velho, both ADE and REF would capture most of the species present. Termites were sampled soil horizons were overlying a plinthic horizon and the REF soil was in five 20 m2 (2 × 10 m) plots near the TSBF monoliths (Figure S1) classified as a Plinthosol. All ADEs were classified as Pretic Clayic by manually digging the soil and looking for termitaria in the soil, Anthrosols, with dark organic-matter-rich surface soil horizons, gen- as well as in the litter and on trees using a modification of the tran- erally >20 cm thick. All soils had greater than 50% clay and clayey sect method of Jones and Eggleton (2000), totaling 50 extra samples texture. from 10 sites. Extra samples for ants were taken only in the forest systems of Iranduba and Belterra. Ants were sampled in 10 pitfall 2.2 | Soil macroinvertebrate sampling traps (300 ml plastic cups) set up as two 5-trap transects on the sides of each 1 ha plot, with 20 m distance between traps (Figure S1), as well as in two traps to the side of each TSBF monolith (distant ~5 m), We performed field sampling in April (Iranduba) and May (Belterra) totaling 20 pitfall traps in each site and 160 samples in total. Each of 2015, and in late February and early March of 2016 (Porto Velho), cup was filled to a third of its volume with water, salt, and detergent at the end of the main rainy season, which is the best time to col- solution. The pitfall traps remained in the field for 48 h. Termites and lect soil macroinvertebrates (Swift & Bignell, 2001). Soil and litter ants were preserved in 80% ethanol and the alcohol changed after macrofauna were collected using the ISO (2011) standard method cleaning the samples within 24 h. All the animals (earthworms, ants, devised by the Tropical Soil Biology and Fertility (TSBF) Program of and termites) were identified to species level or morphospecies level the United Nations Educational, Scientific and Cultural Organization by co-authors as described above. (UNESCO; Anderson & Ingram, 1993), and considered appropriate for evaluating soil macrofauna populations in the tropics. At each one of the 18 sampling sites, five sampling points were located within a 1 ha 2.4 | Soil physical and chemical attributes plot, at the corners and the center of a 60 × 60 m square, resulting in an “X”-shaped sampling design (Figure S1). At each of these points, a After hand-sorting the soil fauna from each TSBF monolith, 2- to soil monolith (25 × 25 cm up to 30 cm depth) was initially delimited 3-kg soil samples were collected from each depth (0–10, 10–20, and with a 10 cm deep steel template, and then divided into surface lit- 20–30 cm) for chemical and soil particle size analysis, and although ter and three 10 cm thick soil layers (0–10, 10–20, and 20–30 cm), analyzed separately, mean values were calculated over 0–30 cm totaling 90 soil monoliths that generated 270 soil layers + 90 lit- depth. The following soil properties were assessed using standard ter samples. Macroinvertebrates (i.e., invertebrates visible to the methodologies (Teixeira et al., 2017): pH (CaCl2); Ca2+, Mg2+, and naked eye and with generally >2 mm body width) were collected in Al3+ (KCl 1 mol L−1); K+ and available P (Mehlich-1); total nitrogen the field by hand-sorting both the soil and surface litter and were (TN) and carbon (TC) by combustion (CNHS). Base saturation, sum immediately fixed in 92% ethanol. Earthworms, ants, and termites of bases (SB) and cation exchange capacity (CEC) were calculated were identified to species or morphologically different morphospe- using standard formulae (Teixeira et al., 2017). Soil texture was de- cies (generally with genus-level assignations) by co-authors SWJ and termined according to the FAO soil texture triangle and the parti- MLCB (earthworms), ACF and RMF (ants), and ANSA (termites), while cle size fractions (% sand, silt, and clay) obtained following standard the remaining macroinvertebrates were sorted into morphospecies methodologies (Teixeira et al., 2017). within higher taxonomic level assignations (e.g., order and/or family). To assess functional differences induced by soil fauna activity in the ADE and REF soils, soil macromorphology samples were taken 2.3 | Additional samples for ecosystem engineers 2 m away from each monolith (Figure S1) using a 10 × 10 × 10 cm metal frame. The collected material was separated into different fractions including living invertebrates, litter, roots, pebbles, pot- As ecosystem engineers (earthworms, termites, and ants) represent tery sherds, charcoal (biochar) fragments, non-aggregated/loose most of the soil macrofauna collected in Amazonian soils (Barros soil, physical aggregates, root-associated aggregates, and fauna- et al., 2006), and we expected them to also be important at the produced aggregates (generally with rounded shapes and darker study sites, we performed additional sampling for earthworms, ter- color than other aggregates) using the method of Velásquez et al. mites, and ants, in order to better estimate their species richness, (2007). Each fraction was oven-dried at 60°C for 24 h and weighed. especially in forest sites, where higher diversity was expected. In This method allows estimating the relative contribution of soil mac- all sampling sites, extra earthworm samples were collected at four rofauna, roots and soil physical processes to soil macroaggregation additional cardinal points of the grid (Figure S1), by hand-sorting and structure, that determine the delivery of important soil-based soil from holes of similar dimensions as the TSBF monoliths and the ecosystem services such as carbon sequestration, water infiltration 6 | DEMETRIO ET al. and availability in the soil, and erosion and flood control (Adhikari & method) and soil properties, we used Generalized Linear Models Hartemink, 2016; Velásquez & Lavelle, 2019). (GLiM) to adjust data to other probability distributions. The best adjustment was Poisson for invertebrate density and Gamma for 2.5 | Treatment of soil fauna data invertebrate density biomass. Soil chemical properties and particle size fractions data were adjusted in Gamma. ANOVA tests were performed with the multcomp package (Hothorn et al., 2008) in R, Density (number of individuals) and biomass of the soil macrofauna adopting a factorial design with the following factors: soil type (ADE surveyed using the TSBF method were extrapolated per square and REF) and LUS (old forests, young forests and agricultural sys- meter considering all depths evaluated. Density and biomass of tems). When factor interactions were significant (p < 0.05), the data immature forms of insects (nymphs and larvae) were grouped in were analyzed comparing the effects of soil type within the LUS and the respective taxonomic group. The following taxonomic groups, the effects of LUS within each soil type. Significant differences were representing 2% or less of total density, were grouped as “Others”: tested using Tukey's test at 95% probability (p < 0.05). Araneae, Hemiptera, Orthoptera, Diptera (larvae), Gastropoda, A Non-Metric Multidimensional Scaling (NMDS) analysis was Dermaptera, Isopoda, Blattaria, Scorpiones, Opiliones, Lepidoptera performed in R (Oksanen et al., 2019) using the densities of earth- (larvae), Thelyphonida, Solifugae, Thysanoptera, Geoplanidae, worms, termites, ants (data from 0 to 10 cm layer) and overall mor- Neuroptera (larvae), Hirudinea, Diplura, Vespidae, and Embioptera. phospecies richness of ecosystem engineers (litter+0–30 cm depth), The earthworms, ants, and termites were also combined into the together with the results of five variables from soil macromorphol- category of ecosystem engineers (Jones et al., 1994; Lavelle et al., ogy (non-aggregated soil, pottery sherds, and fauna-produced, 1997). To calculate the beta (β) diversity index, we removed single- root-associated, and physical aggregates) and 6 variables from soil ton species (species represented by single individuals, i.e., one indi- chemical analyses (pH, Al3+, P, SB, TC, and TN). vidual among all the 8378 individuals collected). 2.6 | Statistical analyses 3 | R E S U LT S 3.1 | ADEs are distinct ecosystems To compare species richness between ADE and REF, we plotted rarefaction and extrapolation curves based on the Chao1 index (Chao, The ADEs at all the sites had higher soil pH (Figure 2a) and were 1984) using the iNEXT package (Hsieh et al., 2016) for total mac- enriched in Ca, Mg, P, and total C compared to REF soils within each roinvertebrate, ant, termite, and earthworm morphospecies diver- LUS (Figure 2b–e), following trends typically observed in ADE sites sity, using the number of TSBF monolith samples as a measure of throughout Amazonia (Lehmann et al., 2003; Sombroek et al., 2003). sampling effort intensity. The same procedure was used for all earth- Significantly lower amounts of exchangeable Al were also found in worm data (9 samples per site), termite data obtained from both the the ADEs (Figure 2f). Soil texture was similar in both ADE and REF 20 m2 plots and TSBF monoliths, and ant data obtained from both soils from each site (Table S1), so the enrichment was not due to pitfall traps and TSBF monoliths. Confidence intervals for rarefac- differences in clay contents, but was the result of ancient anthro- tion and extrapolation curves were obtained by running a bootstrap- pogenic activities (Lehmann et al., 2003; Smith, 1980). Some differ- ping procedure (999 iterations). ences in soil fertility among land-use systems were also observed, We used the betapart package (Baselga & Orme, 2012) in R (R where plots under annual cropping or pasture use in REF soils had Core Team, 2020) to decompose β-diversity (calculated using the higher Ca and Mg contents (due to liming) than the young regenera- Sørensen dissimilarity index) into its Turnover (Simpson index of dis- tion forests (Figure 2b,c), as well as higher K contents and base satu- similarity) and Nestedness components using the species presence/ ration than in both young and old secondary forests (Table S1) due absence (binary data) of all soil and litter macroinvertebrate, ant, to fertilization. Total C and N contents were higher in young regen- termite, and earthworm data from monolith samples. The average eration forests than in agricultural systems and old forests on both β-diversity was calculated to highlight land-use effects, by compar- ADEs and REF soils (Figure 2e; Table S1), owing probably to high or- ing all land-use systems (old forests, young forests, and agriculture) ganic matter deposition in these rapidly regenerating young forests. within each soil type (REF and ADE) and region, thus isolating the We collected 8378 macroinvertebrates in soil monoliths, of 673 land-use effect. The soil type effect was assessed by comparing the different morphospecies (Figure 3) belonging to 26 higher taxa. diversity between REF and ADE soils within each land-use system in Ants were the most diverse group collected (153 spp.), followed by each region. To identify the effect of geographic distance (region ef- spiders (86 spp.), beetles (78 spp.), millipedes (53 spp.), true bugs fect) on species turnover, we also calculated the average β-diversity (42 spp.), earthworms (39 spp.), termites (37 spp.), and cockroaches among the three replicates of each land-use system within each soil (34 spp.) (Figure 3, scientific names of higher taxa can be found in type. Demetrio et al., 2021). Less diverse taxa included isopods (21 spp.), Due to non-normal distribution of both the faunal variables opilionids (21 spp.), centipedes (17 spp.), and snails (17 ssp.) while (i.e., density and biomass of invertebrates collected using the TSBF the less abundant taxa (Others) represented a relatively species-rich | DEMETRIO ET al. 7 F I G U R E 2 Soil chemical properties in the topsoil layer (0–30 cm depth; mean values for the three regions) at the collection sites in Amazonia: (a) pH (in Ca Cl2), (c) exchangeable Ca (cmolc kg−1), (c) exchangeable Mg (cmolc kg−1), (d) available P (mg kg−1), (e) total carbon (g kg−1), and (f) exchangeable Al (cmolc kg−1) in each soil type (REF vs. ADE soils) and land-use system (Secondary forests, Regeneration forests, Agricultural systems). Red asterisks indicate significant differences (p < 0.05) between soil categories (ADE vs. REF) within each land-use system, while different lower-case red letters indicate significant differences among land-use systems within the same soil type. ADE, Amazonian Dark Earth; REF, reference soils. Values shown are median (black line), 1st and 3rd quartiles (box), max/min observations (upper and lower lines), and the outliers (small black circles), when present group, when combined (75 spp.). Furthermore, the number of sin- (Table S2). Furthermore, among the ecosystem engineers collected, gleton species (one individual in the total sample of 8378) was very we found a considerable number of species new to science (>20 high (336 spp.), representing 50% of total macroinvertebrate species earthworm, >20 termite, and >30 ant species) that still must be for- richness (Table S2). mally described. Species richness overall was similar in ADEs (385 spp.) and REF ADEs were home to 52 rare (which include doubletons and mor- (399 spp.) soils, but more species were found in Belterra (314 spp., phospecies with fewer than 10 ind. over all samples) and to 21 non- where two old forests were sampled) than in Porto Velho (238 spp., rare or abundant macroinvertebrate morphospecies (taxa with ≥10 where both forests were young) and Iranduba (218 spp.). More than ind. over all samples) not found in REF soils (Table S2). Interestingly, 50% of all morphospecies were present in old forests, compared within the non-rare/abundant taxa, 16 species (of which seven were with lower and much lower proportions, respectively, in young re- of ants and five were of earthworms) had greater abundance of in- generation forests and agricultural systems (Figure 3n). From all the dividuals in ADEs, while 14 species (half of them ant species) were monoliths, total species richness of ants, earthworms, spiders, bee- more abundant in REF soils (Table S2). Overall, very few species tles, true bugs, cockroaches, and isopods was also fairly similar in were shared between the paired ADE and REF soils at each site, with each soil type (Figure 3a,b,d,e,g–i), but termite richness was much many species unique to each soil type (Figure S2). higher, and centipede and opilionid richness slightly higher, in REF Based on our results from the monolith samples (n = 45 for than in ADE soils (Figure 3c,j,k). On the other hand, richness of both each soil type), estimated richness (i.e., that would have been ob- millipedes and snails was higher in ADE than REF soils (Figure 3f,l), tained with increased sampling effort) for total macroinvertebrates, possibly owing to the higher soil Ca levels found in ADEs (Figure 2b; for ants, and for earthworms (Figure 4a,b,d, respectively) was not Coleman et al., 2004). different between REF and ADE soils. For termites, however, esti- The proportion of exclusive morphospecies was high in both mated richness was three times higher in REF soils (20 vs. 58 spp.; soils: 49% in ADEs and 51% in REF soils (Figure 3n), particularly Figure 4c), and predicted to be attained with 300 samples, that is, for ants (62 spp. were exclusive to ADE, 58 spp. exclusive to REF), more than three times the present sampling effort (90 samples). spiders (39 spp. to ADE, 42 spp. to REF), beetles (31 spp. to ADE, These results were confirmed with the additional samples taken 35 spp. to REF), true bugs (18 spp. to ADE, 21 spp. to REF), and for ants, termites, and earthworms, which showed little difference earthworms (15 spp. to both ADE and to REF; Table S2; Figure 3o). between soil types in the increase in richness of ants and earth- Many more species of termites and opilionids were unique to REF worms compared to the monoliths, but large differences for termites soils (24 and 12 spp., respectively) than to ADE soils (5 and 7 spp., (Figure 4e–g, respectively). The monolith samples (n = 45 for each respectively), while many more species of millipedes and snails were soil type) covered 63% of the estimated richness (up to 100 samples) unique to ADE soils (28 and 10 spp., respectively) than to REF soils of total soil macroinvertebrates and ants in both soil types (Figure (16 and 5 spp., respectively). These trends for ants, earthworms, and S3). Termite richness was slightly better estimated by the monoliths termites remained similar even after singleton species were removed in REF soils (~71%) than in ADEs (~62%), while earthworm richness 8 | DEMETRIO ET al. F I G U R E 3 Morphospecies richness patterns in soil communities found in the monoliths dug at 18 collection sites in Amazonia: Total number of morphospecies of (a) ants, (b) earthworms, (c) termites, (d) spiders, (e) beetles (adults only), (f) millipedes, (g) true bugs, (h) cockroaches, (i) Isopods, (j) opilionids, (k) centipedes, (l) snails, and (m) others (sum of all remaining taxa encountered, including Dermaptera, Diplura, Diptera & Lepidoptera larvae, Embioptera, Geoplanidae, Hirudinea, Neuroptera, Orthoptera, Scorpiones, Solifugae, Thysanoptera, Thelyphonida, and Vespidae), according to soil type (ADE, REF) and land-use systems. The total number of morphospecies of each taxon (a–m) found overall is shown on the top of each graph. (n) Distribution of morphospecies (including singletons) of all macroinvertebrates according to proportion (%) of unique species found in each soil type, region, and land-use system. (o) Numbers of morphospecies of earthworms, termites, and ants observed in both soil categories (blue bars) or uniquely in ADE (black bars) or in REF (red bars) soils, in the different regions (I, B, P) and land-use systems (O, Y, A) across regions. A, agricultural systems; ADE, Amazonian Dark Earth; B, Belterra; I, Iranduba; O, old secondary forests; P, Porto Velho; REF, reference soils; Y, young regeneration forests was relatively well sampled with soil monoliths (especially in ADEs), (Figure 4e–g). Furthermore, increasing the current sampling effort which collected 72%–90% of the estimated species richness (up to could still greatly increase total termite richness, particularly in REF 100 samples) in both soil types (Figure S3). Nonetheless, the use of soils. complementary sampling methods greatly increased the richness The high number of species unique to each soil was reflected in of ants (57–70 additional spp.) and of termites (26–50 additional high β-diversity values and species turnover, ranging from 66% to spp.), and slightly increased that of earthworms (3–4 additional spp.) 87% for all of the soil macroinvertebrates, depending on the region, collected in both soils, revealing a large species pool of these soil LUS and soil type (Table 2). Interestingly, land-use effects on macro- engineers not adequately evaluated using only the TSBF method invertebrate species turnover rates were slightly higher than those | DEMETRIO ET al. 9 F I G U R E 4 Morphospecies rarefaction and extrapolation curves, showing how morphospecies richness increases in both ADE and REF soils depending on sampling intensity (number of samples) for (a) all soil macroinvertebrates, (b) ants, (c) termites, and (d) earthworms considering only soil monolith (TSBF) samples; and for (e) ants collected in pitfall traps + monoliths (TSBF) in old secondary and young regeneration forests in Iranduba and Belterra (Porto Velho data excluded), (f) termites in soil monolith samples + 10 m2 plots in old secondary and young regeneration forests (except one young forest in Porto Velho) and (g) earthworms from all monoliths (n = 9 per plot) samples over all sites. Rarefaction and extrapolation curves were obtained based in Chao1 index. Dark grey and red areas represent 95% confidence intervals (bootstrapping procedure). ADE, Amazonian Dark Earth; REF, Reference soil of soil type, indicating that species turnover was more affected by termites represented 9% to 75% of total macroinvertebrate abun- land-use change than by soil type (Table 2). Similar results were ob- dance, depending on the region and LUS. Ant proportions were less served for earthworms, with much higher turnover rates (0.85 and variable, ranging from 10% to 39% of total abundance. The propor- 0.65 within REF and ADEs, respectively) due to LUS than due to soil tion of ecosystem engineers was significantly higher in Porto Velho type, particularly in old secondary forests. Conversely, soil type had than in Iranduba and Belterra, mainly owing to the higher propor- a greater impact than land use on termite species turnover, while for tion of termites in Porto Velho (Figure 5j), particularly in REF soils. ants, the effect of soil type on species turnover was mainly observed Earthworms were proportionately more abundant in Porto Velho in old secondary forests. The species turnover among regions was (22%) and Iranduba (28%) than in Belterra, where the relative den- also very high, especially for overall macroinvertebrates (all taxa) sity of ants (35%) and non-engineers (43% of total) was greater than and for earthworms in both soils, implying a high number of macro- in the two other regions. invertebrate species (and earthworms) locally endemic to different The proportion of ecosystem engineer individuals found in parts of Amazonia (Table 2). For ants, species turnover was higher in each LUS was not different overall but varied in the ADE soil type, both forest types than in the agricultural systems, implying that ag- where there were proportionally more engineers in the agricultural ricultural systems include a larger proportion of widespread species systems than in the old forests (Figure 5j). Earthworms tended to common to all three sampling regions. be proportionally more important in ADEs while termites were more important in REF soils. Furthermore, engineers were signifi- 3.2 | Ecosystem engineers dominate the soil fauna communities cantly more abundant in REF than ADE soils of all land-use systems (Figure 5h), mainly due to the termite populations that were significantly higher in REF soils of all LUS, with populations over 1000 individuals m−2 (Figure 5a). Meanwhile, with lower total populations, Ecosystem engineers represented on average 72% and 69% of the the earthworm abundance in both agricultural systems and young soil macroinvertebrate individuals found in ADE and REF soils, re- regeneration forests was significantly higher in ADEs than in REF spectively (Figure 5j). In the ADEs, earthworms represented from soils (Figure 5c). Additionally, the abundance of beetles and other 13% to 43% of all individuals collected, while in the REF soils, macroinvertebrates was higher in old forests on ADEs than REF soils 10 | DEMETRIO ET al. TA B L E 2 Effects of region, land-use system (LUS), and soil type (REF and ADE) on β-diversity (without singletons) and species turnover rates of total soil macrofauna (339 morphospecies), ant, termite, and earthworm assemblages. Richness values used for the calculations are from the soil monoliths (TSBF) Partitioned effect Max div. (βSorensen) Turnover (βSimpson dis.) All fauna Region effect Max div. (βSorensen) Turnover (βSimpson dis.) Ants Max div. (βSorensen) Turnover (βSimpson dis.) Termites Max div. (βSorensen) Turnover (βSimpson dis.) Earthworms 1 In REF 0.87 In ADE 0.84 0.87 0.81 0.83 a 0.47 0.76 a 0.93 0.90 0.84 0.84 0.86 0.82 0.84 0.79 0.82 In REF 0.85 0.79 0.90 0.82 0.83 0.67 0.90 0.85 In ADE 0.82 0.74 0.85 0.80 0.80 0.39 0.72 0.65 In O 0.74 0.70 0.86 0.85 0.85 0.79 0.43 0.31 In Y 0.68 0.66 0.82 0.80 0.76 0.68 0.68 0.68 In A 0.74 0.68 0.77 0.61 — — 0.83 0.83 LUS effect 2 Soil effect 3 Abbreviations: A, agricultural systems; ADE, Amazonian Dark Earth; O, old secondary forests; REF, Reference soil; Y, young regeneration forests. a Calculated using only O and Y forest sites. Region: Mean regional effect, presented for each soil type and calculated by averaging all turnovers for each LUS, tested between regions (e.g., old forest at Iranduba vs. old forests at Belterra on REF soil). 1 LUS: Mean effect of all differences in land-use systems, presented for each soil type and within each region, and then averaged across all regions (e.g., both young forests compared with pasture at Porto Velho). 2 Soil: Mean effect of soil type in each land-use system, compared within each region (e.g., old forest in Belterra on ADE compared with old forest in Belterra on REF soil) and then averaged over all regions. 3 and young forests or agricultural systems on ADEs (Figure 5d,g). differences in macrofauna communities between the two (more Also, the abundance of millipedes was higher in young regeneration earthworms in ADEs and more termites in REF soils). Furthermore, forests on ADEs than on REF soils (Figure 5e). the analysis confirmed the role of land-use disturbance or intensifi- Ecosystem engineers represented from 65% to 94% of total cation (the LUS were aligned with the y-axis) as a regulator of eco- soil fauna biomass, with earthworms being the largest component, system engineer biodiversity and the types of aggregates present representing 61%–99% of the engineer biomass and 44%–92% of in the soil, with physical aggregates being more associated with the total macroinvertebrate biomass (Table S3). In both agricultural agricultural systems and fauna-produced aggregates with the more systems and in the young regeneration forests, earthworm biomass conserved forest ecosystems. Pottery sherds were found only in was higher on ADEs than on REF soils. Furthermore, in the young ADE soils, and these are relevant components of ADEs and in their regeneration forests, ecosystem engineer, millipede, other and total classification (Kämpf et al., 2009). macrofauna biomass were also significantly higher on ADEs than on REF soils (Table S3). On the other hand, in all LUS, termite biomass was significantly higher on REF soils than on ADEs. No other higher taxon of soil animals represented more than 16% of the total macro- 3.4 | Modern land use erodes soil biodiversity and function invertebrate biomass in any given soil type or LUS (Table S3). Modern agricultural systems had lower richness of all major soil animal 3.3 | Soil biota influence ADE soil properties taxa (except for true bugs and snails in REF soils and beetles in ADEs; Figure 3) than both forest types (old and young), regardless of soil type (both ADE and REF). Total morphospecies richness at each site Soil macromorphology revealed a significantly higher propor- ranged from 51 (REF, Iranduba) to 91 (ADE, Belterra) in old secondary tion of fauna-produced aggregates in ADEs compared to REF soils forests, from 37 to 80 in young regeneration forests (both ADE sites (Figure 6a), and likewise, for samples from the same LUS, a lower pro- in Porto Velho) and from 18 (maize on ADE in Iranduba) to 44 (soy- portion of non-aggregated soil in ADEs than in REF soils (Figure 6d), bean on ADE in Belterra) in agricultural ecosystems (Figure S2). Over implying important changes in soil structure in ADEs driven by soil all sites 350, 278, and 151 morphospecies of macroinvertebrates were macrofauna bioturbation. found in old and young forests and agricultural systems, respectively, The multivariate analysis (NMDS; Figure 6e) confirmed the im- of which 237, 167, and 63 species were unique to each respective LUS portance of soil fertility variables (particularly Al, sum of bases, (Figure 3n). Removing singleton species, morphospecies richness was and available P contents) in separating ADE and REF soils, and the 135 (old forests), 97 (young forests), and 50 (agricultural systems) in | DEMETRIO ET al. 11 F I G U R E 5 Mean density (Den.; number of individuals m−2) ± standard error of (a) termites, (b) ants, (c) earthworms, (d) beetles (adults + larvae), (e) millipedes, (f) centipedes, (g) others (all the remaining taxa), (h) Ecosystem engineers (i.e., earthworms, ants, and termites), and (i) total macroinvertebrates collected in each land-use system studied, comparing REF and ADE soils. (j) Relative densities (%) of earthworms, termites, ants, and other soil macroinvertebrates (sum of all other taxa) found in the different soil categories (ADE and REF), regions (I, B, P), and land-use systems (O, Y, A). Asterisks indicate significant differences (p < 0.05) in density between soils (ADE vs. REF) within each land-use system, while different lower-case letters indicate significant differences between land-use systems within the same soil type, in the abundance of each taxonomic group (a–g). A, agricultural systems; ADE, Amazonian Dark Earth; B, Belterra; I, Iranduba; O, old secondary forests; P, Porto Velho; REF, reference soils; Y, young regeneration forests ADE soils, and 119 (old forests), 96 (young forests), and 55 (agricul- proportions of physical aggregates (Figure 6b). Root-associated ag- tural systems) in REF soils. Hence, richness was 63% and 55% lower gregates and non-aggregated soil fractions were more abundant in modern agricultural systems compared with old and young forests, in young forests than agricultural systems (Figure 6c), and old for- respectively. This trend was also observed for most of the groups of ests (Figure 6d), respectively, implying important differences in soil soil animals taken individually and was particularly marked (>60% structure dynamics in each LUS, with lower overall biotic contribu- decrease in species richness) for opilionids, centipedes, isopods, and tions to soil functioning in agricultural than in forest systems. cockroaches in both REF and ADE soils, and for earthworms in REF and termites in ADE soils (Figure 3). Species richness decreases in agricultural systems compared to old forests were slightly (but not 4 | DISCUSSION significantly) higher for ADE (66%) than REF (56%) soils. Abundance of predators (centipedes, arachnids, diplurans, ear- Our study found over 670 macroinvertebrate morphospecies in the wigs, scorpions, opilionids, whip scorpions, solifuges, antlion larvae, 18 sites from three Amazonian regions, including at least 70 new leeches, and wasps) and of several individual taxa were also signifi- species of ecosystem engineers. The morphospecies richness ob- cantly lower in agricultural systems (Figure 5) compared with young served at each site (min. 18 in agricultural, max. 91 in old forest) forests (termite and millipedes on ADEs) and old forests (beetles, was within values reported for similar land uses in other Amazonian centipedes, and others on ADEs), or compared with both forest regions (Barros et al., 2006; Mathieu, 2004; Mathieu et al., 2005). systems (earthworms and centipedes on REF soils), highlighting the We also found that although species richness was similar in ADE and negative impact of more intensive ecosystem disturbance on the REF soils, these two habitats harbor very different species pools, populations of these taxa. with few found in both habitats (Figure 3; Figure S2). This high Furthermore, within each soil type, fauna-produced aggregates turnover between sites and number of unique species appears to were more abundant in the old forests compared to the young forests be a prevalent feature of Amazonian rainforest invertebrate com- and agricultural systems (Figure 6a), which had significantly higher munities (Maggia et al., 2021; Mathieu, 2004; Vasconcelos, 2006). 12 | DEMETRIO ET al. Furthermore, although species rarefaction curves were still far from saturation with our current sampling effort, estimated richness showed similar trends, and showcased the wealth of species still to be discovered in both soils (Figure 4). We believe that anthropic soils represent a major gap in the knowledge of Amazonian biodiversity. Soil animals have been poorly represented in taxonomic surveys in Amazonia (Constantino & Acioli, 2006; Franklin & Morais, 2006; James & Brown, 2006; Vasconcelos, 2006), and ADEs had not previously been sampled for soil macrofauna to this extent. Although ADEs occupy only a small fraction (0.1%–3%) of the Amazonian surface area (McMichael et al., 2014; Sombroek et al., 2003), they are scattered throughout the region (Clement et al., 2015; Kern et al., 2017), representing thousands of localized special habitats for species. The high β-diversity values and species turnovers between different ADEs mean that each of these patches may be home to distinctive soil animal communities, including many new species, judging by the number of new ecosystem engineers found. Soil provides chemical and physical support for vegetation and, as millennia of human activities created ADEs in the Amazon, patches with higher amounts of nutrients and organic resources were generated throughout a matrix of poorer soils (Kern et al., 2017; Macedo et al., 2019). The formation processes and human management of these soils result in distinct plant and microbial communities (Brossi et al., 2014; Clement et al., 2015; Levis et al., 2018; Taketani & Tsai, 2010), that are a result of disturbance, soil enrichment, and selection processes (both natural and humandriven). Here we show that current soil animal abundance and diversity also reflect the impact of these ancient anthropogenic activities. The ADEs developed a different pool of species compared with REF soils. The former soils tend to favor more animals that recycle organic matter and flourish with higher pH and soil F I G U R E 6 Macromorphological aggregate fractions (%) and their relationships to various soil attributes (0–10 cm layer) in two different Amazonian soils (ADE, Amazonian Dark Earth; REF, non-anthropogenic reference soils) and three different land-use systems (A, agricultural systems; O, old secondary forests; Y, young regeneration forests); (a) fauna-produced aggregates (FA), (b) physical aggregates (PA), (c) root aggregates (RA), (d) nonmacroaggregated loose soil particles and unidentified aggregates less than 5 mm in size (NAS). Values shown are relative mean (%) ± standard error. Asterisks indicate significant differences (p < 0.05) between soil categories within each land-use system, while different lower-case letters indicate significant differences between land-use systems within the same soil type. (e) Non-metric Multidimensional Scaling (NMDS) of soil macroinvertebrate data, combined with soil macromorphology features and soil chemical properties: Blue letters: macromorphological fractions (FA, faunaproduced aggregates; NAS, non-aggregated soil; PA, physical aggregates; Pot, Pottery sherds; RA, root-associated aggregates). Black letters: density (no. ind. m−2) of ants (An), termites (Te) and earthworms (Ew), and overall ecosystem engineer morphospecies richness (Es). Red letters: soil chemical properties (Al, exchangeable aluminum; CEC, cation exchange capacity; P, available phosphorus, pH; SB, sum of bases; TC, total carbon) Ca, like earthworms and millipedes, while the latter favor termites, which are particularly sensitive to deforestation and changes in soil moisture and physical conditions (Dambros et al., 2013; de Souza & Brown, 1994; Duran-Bautista, Muñoz Chilatra, et al., 2020; Eggleton et al., 1996). Similar microenvironmental characteristics of soil matrix and overlying vegetation probably have, and continue to influence soil fauna community composition in other anthropic soils in various regions of the world, such as in West Africa, Europe, and Central America (Macphail et al., 2017; Solomon et al., 2016; Wiedner et al., 2014). However, further elucidation of the pathways to changed community composition (and possibly species diversification) in ADEs and other anthropic soils would require expanding microbial and invertebrate biodiversity inventories. The functional particularities observed in biotic communities of ADEs also mean that ecosystem functioning could be different in these soils, which could imply differences in their ecosystem services to humans, as observed in other human-altered landscapes in Amazonia (Marichal et al., 2014; Rodríguez et al., 2021; Velásquez & Lavelle, 2019). Although relationships between the changes in macrofauna communities and soil aggregation, on the one hand, and ecosystem | DEMETRIO ET al. 13 service delivery, on the other, have been mostly indirect (correlation special attention and management, to discover and protect their bi- rather than causation), it is well known that larger earthworm popula- ological resources and promote more sustainable uses of Amazonian tions and improved soil structure owing mainly to fauna-produced ag- soils (Glaser, 2007). gregates (as occurs in ADE) can alter soil hydraulic properties (Alegre et al., 1996; Hallaire et al., 2000), primary productivity (Brown et al., AC K N OW L E D G E M E N T S 1999; Pashanasi et al., 1996), litter decomposition, and nutrient cy- The study was supported by the Newton Fund and Fundação cling (Lavelle et al., 2006) as well as pedogenetic processes (Cunha Araucária et al., 2016; Macedo et al., 2017), and could help stabilize organic N000323/1), Natural Environment Research Council (NERC) UK carbon in these soils (Cunha et al., 2016; Ponge et al., 2006). On the (grant No. NE/M017656/1), a European Union Horizon 2020 Marie- other hand, larger termite populations in REF soils could be contribut- Curie fellowship to LC (MSCA-IF-2014-GF-660378), and DWGS (No. ing to ecosystem services as well (Duran-Bautista, Armbrecht, et al., 796877), by CAPES scholarships to WCD, ACC, TF, RFS, AF, LM, HSN, 2020), particularly in old forests, where fauna aggregates were also TS, AM, and RSM (PVE A115/2013), Araucaria Foundation scholar- abundant. The links between soil fauna populations, land use, and ships to LM, AS, ACC, and ES, Post-doctoral fellowships to DWGS ecosystems service delivery merit further attention, both in forested (NERC grant NE/M017656/1) and ES (CNPq No. 150748/2014- 0, and agriculturally managed soils, particularly in ADEs. 165702/2015- 0), PEER (Partnerships for Enhanced Engagement (grant Nos. 45166.460.32093.02022015, NE/ As archeological sites, ADEs are protected by Brazilian law (Lei in Research Science Program) NAS/USAID award number AID- N o 3.924 de 26 de Julho; Brasil, 1961), but throughout Amazonia OAA-A-11- 0001—project 3-188 to RMF and by CNPq grants, they are actively sought out and intensively used for agricultural scholarships, and fellowships to ACF, GGB, RF, SWJ, CRC, EGN, and horticultural purposes (Fraser et al., 2011; Junqueira et al., and PL (Nos. 140260/2016-1, 307486/2013-3, 302462/2016-3, 2016; Kern et al., 2017). Intensive annual cropping and extensive 401824/2013-6, 310690/2017- 0, 303477/2018- 0, 307179/2013-3, livestock production represent a threat to soil macrofauna popu- and 400533/2014-6). We thank INPA, UFOPA, Embrapa Rondônia, lations, both in REF and in ADE soils. Macroinvertebrate diversity Embrapa Amazônia Ocidental, and Embrapa Amazônia Oriental in both soils decreased dramatically with increasing environmen- and their staff for logistical support and the farmers for access to tal disturbance (Figures 3 and 5), and negative impacts on some and permission to sample on their properties. Sampling permit No. macroinvertebrate populations were higher in ADE than in REF 18131-6 for Tapajós National Forest was granted by ICMBio. soils. Modern human activity is often associated with negative environmental impacts in the Amazon (Decaëns et al., 2018; Franco C O N FL I C T O F I N T E R E S T et al., 2018), but on the other hand, the Pre- Columbian histori- The authors declare no conflict of interests. cal human footprints associated with ADE formation processes and their long-term traditional use appear to have “positive” ef- DATA AVA I L A B I L I T Y S TAT E M E N T fects on the Amazonian ecosystem (Balée, 2010). For instance, Demetrio, Wilian et al. (2020), A “Dirty” Footprint: Biodiversity we found that old forests on ADEs were the most diverse LUS in Amazonian Anthropogenic Soils, Dryad, Dataset https://doi. in terms of total soil macroinvertebrate morphospecies, and have org/10.5061/dryad.3tx95x6cc (in review process). also been shown to contain numerous useful tree and palm species (Levis et al., 2017, 2018). Soil invertebrates are known to display high endemism (Lavelle & ORCID Wilian C. Demetrio Lapied, 2003), and hence high β-diversity values, mainly due to their George G. Brown low dispersal ability (Wu et al., 2011). Still, the high turnover rates Luís Cunha https://orcid.org/0000-0003-0052-4587 https://orcid.org/0000-0001-9550-6909 https://orcid.org/0000-0002-5870-2537 between communities of ADE and REF soils suggest that ADEs may represent refuges for large numbers of specialist species that have REFERENCES been overlooked in previous work in the region (Barros et al., 2006; Adhikari, K., & Hartemink, A. E. (2016). Linking soils to ecosystem services — A global review. Geoderma, 262, 101–111. https://doi. org/10.1016/j.geoderma.2015.08.009 Alegre, J. C., Pashanasi, B., & Lavelle, P. (1996). Dynamics of soil physical properties in a low input agricultural system inoculated with the earthworm Pontoscolex corethrurus in the amazon region of Peru. Soil Science Society of America Journal, 60, 1522–1529. Alho, C. F. B. V., Samuel-Rosa, A., Martins, G. 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