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Over the last few years, the recovery and the analyses of genomes of ancient modern humans, Neandertals, and Denisovans have changed our understanding of the origins, the movements, and the relatedness of archaic and modern human populations. How- ever, in many cases endogenous DNA represents such a small fraction of the DNA extracted from specimens that sequencing of the complete ancient genomes is economically infeasible. Thus, to date, only three Neandertal genomes have been sequenced to high coverage [1-3]. Even though Neandertal genome sequences of low coverage [4] can be used to reconstruct various aspects of Neandertal genetic history, many analyses, for example estimation of population size and levels of inbreeding, rely on the reliable diploid genotypes. Recent studies have shown that certain skeletal elements, such as the inner part of the petrous bone and the ce- mentum layer in teeth [5 and references therein], may preserve DNA better over time. There is also evidence that the preservation of endogenous DNA may vary substantially even within a few millimeters distance in a single specimen [2, 4]. Due to the value and scarcity of ancient hominin remains, it is critical that the smallest possible amount of destructive sampling is involved in the recovery of genetic material. A usual sampling strategy typically involves taking around 50 mg of powder from a single location of a given bone or tooth. We investigated here whether taking multiple smaller samples in a step-wise manner of the Neandertal specimens from the Mezmaiskaya Cave in Russia and the Troisième caverne of Goyet in Belgium may improve the yield of an- cient human DNA. We removed between 8.5 and 27.2 mg of bone powder from a Mezmaiskaya 1 rib fragment, between 2.5 and 35.1 mg from a Mezmaiskaya 2 skull fragment, and between 5.8 and 53.8 mg from the Goyet Q56-1 femur fragment, amounting to between 15 and 38 powder subsets per specimen and an average input of 16.6 mg of powder per extraction. Importantly, to minimize the impact of contamination, we treated each powder aliquot with 0.5\% sodium hypochlorite solution prior to DNA extraction. The DNA extracts from the same specimen varied by several orders of magnitude in their proportion of endogenous DNA (between 0.07\% and 54.7\%), their content of nuclear genomes (between 0.01 and 78-fold coverage), as well as in the levels of present-day human contamination (0.2-50.3\%). There was no significant correlation between the amount of powder used for the extraction and the overall amount of the endogenous DNA or the levels of present-day human DNA contamination. Thus, these results indicate that ancient DNA preservation varies greatly within one specimen and that the removal of multiple, small sub-samples instead of one larger sample, here coupled with a decontamination procedure, can drastically improve the likelihood of isolating large enough amounts of DNA to make whole genome sequencing feasible. This approach allowed us to identify extracts with exceptionally high endogenous DNA content and low levels of present-day human DNA contamination (2\%), enabling us to generate three additional high-coverage Neandertal genomes. The high-quality genome sequences of multiple Neandertals form a unique reference resource for the scientific community and are valuable for analyses that require reliable diploid genotypes and haplotype information. For example, these data open new opportunities to investigate Neandertal population history, to identify genetic variants that arose uniquely on the Neandertal lineage and might have changed through time, and to determine those that may underlie archaic-specific traits or adaptations.

The main excavations at the Troisième caverne of Goyet in Belgium were conducted by Edouard Dupont in 1868 who identified Palaeolithic human occupations later attributed to the Middle and Upper Palaeolithic. These are represented by an archaeologi- cal record that spans the Mousterian, Lincombian-Ranisian-Jerzmanowician, Aurignacian, Gravettian, and Magdalenian, and then extends into the Neolithic and historic periods. Due to the lack of detailed documentation of the excavated materials, their asso- ciation to a specific chronocultural context has been challenging. Morphometric and taphonomic analyses, combined with direct radiocarbon dating as well as isotopic and genetic analyses, were used to assign human remains to either late Neanderthals or an- cient modern humans from different chronocultural groups. In 2016 the first palaeogenetic investigation of Neanderthal specimens from Goyet was published [1]. Taxonomic assignment was confirmed by performing hybridization capture of the mitochondrial DNA (mtDNA) and later inspecting diagnostic mutations at nucleotide positions that distinguish modern humans from Nean- derthals. Moreover, a phylogenetic reconstruction placed seven nearly complete mtDNA sequences from Goyet within the diver- sity of late Neanderthal mtDNA. An around two-fold coverage nuclear genome was later sequenced from one of those individuals (Goyet Q56-1) [2], revealing a high genetic similarity to other late Neanderthals that is well correlated to their geographical dis- tance. Analyzing modern human remains retrieved at Goyet, mtDNA genomes were initially reported for two specimens directly dated to the Aurignacian, five to the Gravettian, and one to the Magdalenian [3]. Aurignacian-related individuals were particu- larly intriguing as they were found to carry mtDNA haplogroup M, which is almost entirely absent in present-day Europeans. For Gravettian- to Magdalenian-related individuals, the shift from U2/U5 to U8 haplogroups was detected locally - as in other regions of Central Europe - likely influenced by the genetic bottleneck during the Last Glacial Maximum (LGM). Furthermore, nuclear sequences of five modern human individuals from Goyet were produced through genome-wide targeted enrichment [4] revealing local replacement between Aurignacian- and Gravettian-related populations. However, the genetic component associated with a 35,000-year-old individual (Goyet Q116-1) reappeared after the LGM, first in Spain and then in other European regions includ- ing in a Magdalenian-related individual from Goyet (Goyet Q-2). This individual was later found to be the best proxy for a genetic component that was largely displaced in Europe from around 14,000 years ago onwards while surviving in high proportion among Mesolithic individuals from Iberia [5]. Here we present new palaeogenetic data of Neanderthal and modern human individuals from this iconic site. First, we expand the molecular taxonomic identifications with three additional Neanderthal specimens and reconstruct their partial mtDNA genomes. Those confirm the general picture of a limited genetic diversity for late Neanderthals, which is also apparent among the Goyet Neanderthals. Second, working on modern human remains, we produced new mtDNA and nuclear data from four Gravettian specimens. They belong to mtDNA haplogroups U2 and U5, further extending the observa- tion of both mtDNA types being largely present in pre-LGM Europe. Moreover, their nuclear genomes provide additional evidence for the genetic affinity between Gravettian-related groups across Europe, from the present-day regions of the Czech Republic to Belgium and Southern Italy. In conclusion, the deep temporal range covered by the human remains from the Troisième caverne of Goyet provides the unique opportunity to describe within a single archaeological site the major genetic transformations that took place in Europe throughout the Middle and Upper Palaeolithic.

Trou Magrite is a cave site located at Pont-à-Lesse in the Lesse Valley, commune of Dinant, Belgium. It has been known since E. Dupont conducted excavations at the site in 1867 [1]. The most recent fieldwork was done by L. Straus and M. Otte in 1991-92 [2]. Trou Magrite yielded rich lithic assemblages, osseous artifacts, mobiliary art, and numerous faunal remains. Several human re- mains were also recovered and identified as Palaeolithic humans by E. Dupont but have been only partially published thus far. The archaeological record covers a broad time range spanning from the Middle and Upper Palaeolithic to the Mesolithic, Neolithic, and Iron Age. An important Middle Palaeolithic collection is present, probably representing several occupation phases during the Late Pleistocene [2]. Unfortunately, although E. Dupont conducted excavations that can be characterized as modern for that time, the materials from the different so-called “fauna-bearing levels” that he defined in the field were mixed post-excavation [3]. In 2015, we initiated a multidisciplinary re-assessment of the human and faunal collections from Trou Magrite in order to update the inven- tory of human remains already identified, check for the presence of human remains that may have been previously overlooked, and verify their chronocultural context. We revised the already known human collection, conducted a systematic sorting of the faunal material, and combined the use of morphometrics, taphonomy, stable isotopes, dating, and genetic analyses to perform taxonomic and chronocultural identifications. Here we present two previously unidentified Neandertal fossils that we isolated from the Trou Magrite faunal material excavated by E. Dupont in the 19th century. They represent two different individuals: an adult/adolescent, represented by an upper right permanent canine, and a neonate, represented by the diaphysis of a left femur. Whereas no endoge- nous DNA was recovered from the tooth, the palaeogenetic analyses of the neonate femur confirmed its Neandertal status and indicate its sex to be male. We will present the biological characteristics and mitochondrial DNA phylogenetic position of the Trou Magrite Neandertals, in particular with regard to the other Northern European Neandertals. Our project adds Trou Magrite to the list of Belgian sites that have yielded Neandertal fossils and helps to emphasize the importance of the Mosan Basin in Neandertal studies.

Subsistence strategies are key paleoecological features of Paleolithic hunter-gatherers and their deeper understanding provides crit- ical insights into essential aspects of human evolution. In this study, we discuss new collagen stable isotopic values (C, N, S) rep- resenting seven Gravettian individuals from the Troisième caverne of Goyet in Belgium. The dietary strategies of the Gravettian humans from Goyet are in line with the general trends observed among Western European Gravettian populations. These pop- ulations show both a low intake of mammoth and a high consumption of other terrestrial mammals as well as aquatic resources, such as at the sites Arene Candide and La Rochette. This is different for more eastern Gravettian hunter-gatherers, for example in Kostenki, Brno-Francouzska, Mal’ta, Předmostí, and Dolní Věstonice where the dietary contribution of mammoth meat was sig- nificantly higher. The stable isotopic data of the Gravettian humans from Goyet indicate that their dietary ecology was essentially based on terrestrial resources like reindeer, horse, and, to a lesser extent, mammoth. However, they yielded δ15N values that are substantially lower than those of the earlier modern humans and Neandertals from the same site [1-2]. We hypothesize that the Gravettian humans had much less mammoth in their diet than all earlier humans from the same region. It was previously shown that in northwestern Europe a decline of mammoth, a key prey species, could already be detected at the onset of the Upper Paleolithic [2]. This trend appears to continue into the Gravettian, despite the persistence of the typical mammoth ecological niche, which is represented by a grassland with high δ15N values. Interestingly, through isotopic analysis, we are able to track the spread of the horse from the local ecosystem (represented by specimens from Walou Cave, Belgium) into this niche now under-occupied by the mammoth. Radiocarbon dates obtained from several mammoth skeletal remains from the Troisième caverne of Goyet showed that this megaherbivore was indeed part of the ecosystem during pre-LGM periods. However, from the Gravettian in Goyet and the surrounding region we have only one mammoth specimen represented by a long bone, and interestingly, its sulphur isotopic signal indicates that this individual was not of local origin. We propose that the local mammoth population was under intensive hunting pressure or may even have been no longer present in the region. Instead, single individuals from other regions may have made it into the area and ended up as prey animals. While the δ15N values of all Goyet Gravettian humans are relatively homogeneous, their δ13C values are variable. This indicates significant dietary differences among the seven individuals, an observation that has not been described before for hunter-gatherers pre-dating the Gravettian. The human δ34S values also support substantial differences in life mobility history between different individuals, which were not observed for the Goyet Neandertals. The result that different mem- bers of the same chrono-group had various individual mobility histories has implications for land use procurement strategies of those hunter-gatherer groups. In conclusion, our new isotopic results demonstrate a broad ecological flexibility among Gravettian humans, which can be seen in different human ecosystem interactions across Europe. The Goyet individuals contribute substan- tially to a more complete understanding of hunter-gatherer’s ecology during this particular phase of the European Late Pleistocene. Our study shows that the Gravettian cannot be depicted as a uniform entity from an ecological perspective. It instead indicates that during this period, and not earlier, both inter- and intra-group diversity in subsistence strategies can be tracked through stable isotopic analysis.

Large tropical trees are rightly perceived as supporting a plethora of organisms. However, baseline data about the variety of taxa coexisting on single large tropical trees are lacking and prevent a full understanding of both the magnitude of biodiversity and the complexity of interactions among organisms in tropical rainforests. The two main aims of the research program “Life on Trees” (LOT) are (1) to establish baseline knowledge on the number of eukaryote species supported/hosted by the above-ground part of a single tropical tree and (2) to understand how these communities of organisms are assembled and distributed on or inside the tree. To achieve the first goal, we integrated a set of 36 methods for comprehensively sampling eukaryotes (plants, fungi, animals, protists) present on a tropical tree. The resulting LOT protocol was conceived and implemented during projects in the Andean Amazon region and is proposed here as a guideline for future projects of a similar nature. To address the second objective, we evaluated the microclimatic differences between tree zones and tested state-of-the-art terrestrial laser scanning (TLS) and positioning technologies incorporating satellite and fixed base station signals (dGNSS). A marked variation in temperature and relative humidity was detected along a 6-zones Johansson scheme, a tree structure subdivision system commonly used to study the stratification of epiphytic plants. Samples were collected from these six zones, including three along the trunk and three in the canopy. To better understand how different tree components (e.g., bark, leaves, fruits, flowers, dead wood) contribute to overall tree biodiversity, we categorized observations into communities based on Johansson zones and microhabitats. TLS was an essential aid in understanding the complex tree architecture. By contrast, the accuracy of positioning samples in the tree with dGNSS was low. Comprehensively sampling the biota of individual trees offers an alternative to assessing the biodiversity of fewer groups of organisms at the forest scale. Large old tropical trees provide a wealth of microhabitats that encompass a wide range of ecological conditions, thereby capturing a broad spectrum of biodiversity.

We present new Gabonese locality records, ecological data or unpublished museum material for Crocodylus niloticus (Crocodylidae), Trionyx triunguis (Trionychidae), Agama lebretoni (Agamidae), Hemidactylus fasciatus and H. mabouia (Gekkonidae), Gastropholis echinata (Lacertidae), Trachylepis albilabris (Scincidae), Afrotyphlops angolensis (Typhlopidae), Dipsadoboa viridis, Hapsidophrys smaragdinus, Toxicodryas pulverulenta (Colubridae), Naja melanoleuca (Elapidae), Lamprophis olivaceus, Psammophis cf. phillipsii (Lamprophiidae), Natriciteres fuliginoides (Natricidae), Causus lichtensteinii and C. maculatus (Viperidae). We document predation cases by Hapsidophrys smaragdinus on Hemidactylus mabouia and Trachylepis albilabris, by Naja melanoleuca on Sclerophrys regularis (Anura: Bufonidae) and by Psammophis cf. phillipsii on Phrynobatrachus auritus (Anura: Phrynobatrachidae), and consumption of Arius latiscutatus (Siluriformes: Ariidae) and Tragelaphus spekii (Cetartiodactyla: Bovidae) by Crocodylus niloticus. We add one, two and one snake species, respectively, to Estuaire, Moyen-Ogooué and Nyanga provinces’ reptile lists. We add four reptile species to the list for Wonga-Wongué Presidential Reserve. We refer all records of Agama agama in Gabon to A. picticauda.