Periodic Reporting for period 2 - HOOKaWORM (Hook a Worm to Catch a Man: Tracking Historical and Recent Human Settlement, Land use and Migration in Neotropical Rainforests using Ecosystem Engineers) Reporting period: 2017-05-01 to 2018-04-30 Summary of the context and overall objectives of the project During the Holocene, humans have greatly altered global patterns of biodiversity and ecosystem dynamics. Numerous examples exist of ancient land-use systems in human-transformed landscapes, supporting vast numbers of people, and greatly modifying the forest cover. One of these are the Amazonian Dark Earths (ADEs): highly nutrient-enriched soils with an outstanding capacity for carbon sequestering, agricultural production and resilience to desertification. Surprisingly, the contribution of soil biota has been severely ignored in these soils, and little is known of their biodiversity. Therefore, the applicant’s intention was to contribute to the knowledge of soil animal biodiversity and its relationship with soil fertility and land use changes in a mega-diverse biome (Amazonia). Promoting the interdisciplinary connection between archaeological data, soil ecology and genomics, the applicant investigates both the relationship of ADEs to the associated extant biodiversity, revealing details of past and current anthropic impact on the natural surroundings, as well as new clues regarding human-settlement dynamics over a large part of Brazil. This is achieved by integrating the state-of-the-art methods in invertebrate taxonomy/morphology, molecular genetics, and computational analyses with incorporation of pre-existent and new ecological, anthropological/archaeological and environmental metadata.This is being pursued through a three-tier approach with three distinct but complementary objectives, compartmentalized into individual workpackages. Therefore, the main objectives of the project are:1st objective: Assessment of the extent of the relation between soil biodiversity and human settlements associated with Amazonian Dark Earths (ADEs)2nd Objective: Tracking recent and historical (i.e. pre-Columbian) land use changes using soil biodiversity assemblages3rd Objective: Tracking Amerindian migration impacts on genetic diversity from western Amazonia to Southern Brazil using a peregrine earthworm species Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Ten sites (ADE & adjacent reference soil) were sampled for soil fauna, including the assessment of the main ecosystem engineer populations (earthworms, ants, and termites) using ISO standard method (n= five 25 x 25 x 30 cm deep soil monoliths per site). Soil chemical parameters and macromorphology were assessed on samples from the 0-30 cm soil layer. Study sites included old-growth forests, young forests, and agricultural systems in the regions of Belterra (PA), Iranduba (AM), and Porto Velho (RO). Qualitative sampling (~80 sites) along a transect from western Amazonia (Rondonia) to Southern Brazil (Parana) and to North (Roraima) was also accomplished. Main results achieved so far:ADE soil species composition is staggeringly different when compared to adjacent soils with a large number of exclusive representatives in ADE, with some newly discovered species. A higher proportion of ecosystem engineers was found in ADEs, closely associated to the presence of bio-aggregates, potsherds and to the soil’s unique physical and chemical properties. Species richness was also lower in agricultural sites and higher in old-growth forest. In total, 530 specimens were selected for DNA analysis, of which the COI was amplified and sequenced, obtaining a successful amplification in ~60% of the cases. These sequences generated a total of 45 MOTUs (Figure 5). The intra-MOTU mean divergence varied between 0 to 3% and between-MOTU from higher than 16%, considering a genetic distance of at least 15% for separation of MOTUs, the identified “barcode gap” (3% - 15%) corresponded to the intervals of divergence generally observed between well-established species in well-known earthworm families, such as Lumbricidae.The richness of MOTUs in ADEs (24 spp.) was lower than in adjacent reference soils (32 spp.). The number of MOTUs found exclusively in only one of the soils was quite large: 21 in the adjacent reference sites (47% of the total) and 13 spp. in TPIs (29% of the total). The specimens were categorized morphologically into four families (Glossoscolecidae, Rhinodrilidae, Ocnerodrilidae and Acanthodrilidae) and tentatively in several genera, including Righiodrilus, Atatina, Pontoscolex, Martiodrilus, Neogaster and Dichogaster. Among the morphotypes found, most are unknown species, without correspondence in open reference databases (e.g. GenBank/NCBI) or with other species known morphologically. Only three could be identified at the species level: Pontoscolex corethrurus (MOTU5), Dichogaster andina (MOTU6) and Dichogaster modiglianii (MOTU9). These three species are well known to invade and inhabit human disturbed landscapes. Geographically, there were nine MOTUs exclusive found in Porto Velho, 18 in Belterra and 21 in Iranduba (Figure 3B). This indicates that composition is mainly constituted by species from regional groups, which is not surprising as earthworms are known for their limited dispersal capacity, and because most of these species are specialists, which makes them sensitive to environmental filters.Results suggest that the unique enriched environment of ADEs became so conspicuously different from adjacent land that it developed its own characteristic fauna. We argued that the human impact in this ecosystem is broader than ever before estimated. This is true for the ecosystem engineers which seem to be a main component of ADEs, representing more than half of the total macrofauna and >80% of total biomass. They therefore contribute significantly to the functioning and engineering of these human-modified ecosystems. It is accepted that the ancient human societies that occupied the region adopted sedentary habits and the presence of exclusive representative ADEs species across the sites may reflect these ancient human dynamics, in particular, exchange of crops. This process of human-mediated transport would explain the distributions that are observed in some of the widely-distributed soil fauna in Brazil. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Succinctly, our results suggest that ADEs appear to constitute a highly unique soil ecosystem, which is distinct from the adjacent soil. As ADEs are anthropogenic in nature, we argue that the impact of humans in the Amazon is even more considerable than previously estimated. Our results show that the study of soil organisms in impacted versus non-impacted soils provide promise for better understanding the effect of humans (both modern and ancient) in this landscape and the extent of “gardening”. This has profound implications for our understanding of ecological gradients, biodiversity distribution, and ecosystem functioning, and must be considered in further initiatives for conservation and management of the Amazonia Biome. Earthworms in particular, display a number of properties that may make them an excellent bioproxy for ancient human dispersal events.It is important to reveal, that most of the work could not be possible without the established wide collaborative network Terra Preta de Índio Network (TPInet) (TPI Network: http://tpinet.org) implemented during the first year of the project. The available expertise and human resources (associated with the different project workpackages according to various expertise) available in the TPInet is mainly characterized by a multidisciplinary nature, that tends to work by a dynamic interdisciplinary interaction.