How to extract and analyze pollen from internal organs and exoskeletons of fossil insects?

This protocol explains how to extract pollen from fossil insects with subsequent descriptions of pollen treatment. We also describe how to document morphological and ultrastructural features with light-microscopy and electron microscopy. It enables a taxonomic assignment of pollen that can be used to interpret flower-insect interactions, foraging and feeding behavior of insects, and the paleoenvironment. The protocol is limited by the state of the fossil, the presence/absence of pollen on fossil specimens, and the availability of extant pollen for comparison. For complete details on the use and execution of this protocol, please refer to Wappler et al. (2015), Ulrich and Grímsson (2020), and Wedmann et al. (2021).

Species distribution models advance our knowledge of the Neanderthals' paleoecology on the Iranian Plateau.

Neanderthals (Homo neanderthalensis) were distributed across a vast region from Europe to western and Central Asia. The Neanderthals' paleoecology and distribution has been extensively studied in Europe where the species originated. However, very little is known about their paleoecology in south-western Asia. Here, we employed species distribution modelling and 45 Middle Palaeolithic (c. 200,000-40,000 years BCE) sites location associated with fossil and/or lithic artefacts made by the Neanderthals to examine the expansion of the Neanderthals on the Iranian Plateau in south-western Asia. We estimated the niche overlap between Neanderthals and wild goat, wild sheep and Persian gazelle by modelling their past distribution using 200, 143 and 110 occurrence records respectively. The results show that Neanderthals had highest niche overlap with wild goat in the study area. This analysis revealed that the most suitable Neanderthals' habitats in south-western Asia were located in the Zagros Mountains stretches from north-western and western and some isolated patches in the central parts of the Iranian Plateau. The annual precipitation and maximum temperature of the warmest month were the most important predictor of the species' distribution. This finding shows that the southern edge of the Neanderthals distribution was limited by warm summer. Our results provide important information for future field investigations and excavations in the area.

Aboriginal artefacts on the continental shelf reveal ancient drowned cultural landscapes in northwest Australia.

This article reports Australia's first confirmed ancient underwater archaeological sites from the continental shelf, located off the Murujuga coastline in north-western Australia. Details on two underwater sites are reported: Cape Bruguieres, comprising > 260 recorded lithic artefacts at depths down to -2.4 m below sea level, and Flying Foam Passage where the find spot is associated with a submerged freshwater spring at -14 m. The sites were discovered through a purposeful research strategy designed to identify underwater targets, using an iterative process incorporating a variety of aerial and underwater remote sensing techniques and diver investigation within a predictive framework to map the submerged landscape within a depth range of 0-20 m. The condition and context of the lithic artefacts are analysed in order to unravel their depositional and taphonomic history and to corroborate their in situ position on a pre-inundation land surface, taking account of known geomorphological and climatic processes including cyclone activity that could have caused displacement and transportation from adjacent coasts. Geomorphological data and radiometric dates establish the chronological limits of the sites and demonstrate that they cannot be later than 7000 cal BP and 8500 cal BP respectively, based on the dates when they were finally submerged by sea-level rise. Comparison of underwater and onshore lithic assemblages shows differences that are consistent with this chronological interpretation. This article sets a foundation for the research strategies and technologies needed to identify archaeological targets at greater depth on the Australian continental shelf and elsewhere, building on the results presented. Emphasis is also placed on the need for legislation to better protect and manage underwater cultural heritage on the 2 million square kilometres of drowned landscapes that were once available for occupation in Australia, and where a major part of its human history must lie waiting to be discovered.

Shelter in Smolen III - A unique example of stratified Holocene clastic cave sediments in Central Europe, a lithostratigraphic stratotype and a record of regional paleoecology.

A cave site Shelter in Smolen III (southern Poland) contains an approximately 2-m-thick stratified sequence of Upper Pleistocene and Holocene clastic sediments, unique for Central Europe. The sequence contents abundant fossil fauna, including mollusk, rodent and bat remains. The cave sites with long profiles of subfossil fauna present a great value for reconstructions of regional terrestrial paleoenvironment. We explore the stratigraphy of this site through analyses of the lithology and geochemistry of sediments, radiocarbon dating of faunal and human remains and charcoals, and archaeological study, as well as the paleoecology derived from the taxonomic composition of fossil faunal assemblages. Our data show that the entire period of the Holocene is recorded in the rockshelter, which makes that site an exceptional and highly valuable case. We present paleoenvironmental reconstructions of regional importance, and we propose to regard Shelter in Smolen III as a regional stratigraphic stratotype of Holocene clastic cave sediments.

Coincidence of photic zone euxinia and impoverishment of arthropods in the aftermath of the Frasnian-Famennian biotic crisis.

The lowermost Famennian deposits of the Kowala quarry (Holy Cross Mountains, Poland) are becoming famous for their rich fossil content such as their abundant phosphatized arthropod remains (mostly thylacocephalans). Here, for the first time, palaeontological and geochemical data were integrated to document abundance and diversity patterns in the context of palaeoenvironmental changes. During deposition, the generally oxic to suboxic conditions were interrupted at least twice by the onset of photic zone euxinia (PZE). Previously, PZE was considered as essential in preserving phosphatised fossils from, e.g., the famous Gogo Formation, Australia. Here, we show, however, that during PZE, the abundance of arthropods drastically dropped. The phosphorous content during PZE was also very low in comparison to that from oxic-suboxic intervals where arthropods are the most abundant. As phosphorous is essential for phosphatisation but also tends to flux off the sediment during bottom water anoxia, we propose that the PZE in such a case does not promote the fossilisation of the arthropods but instead leads to their impoverishment and non-preservation. Thus, the PZE conditions with anoxic bottom waters cannot be presumed as universal for exceptional fossil preservation by phosphatisation, and caution must be paid when interpreting the fossil abundance on the background of redox conditions.

Evidence of congenital block vertebra in Pleistocene Cave Bear (Ursus spelaeus) from Cueva de Guantes (Palencia, Spain).

OBJECTIVE: This work provides a detailed description and differential diagnosis of a Pleistocene cave bear (Ursus spelaeus). MATERIALS: The specimen was recovered at the Cueva de Guantes archaeo- paleontological site, located in the North of the Iberian Peninsula and dated to more than 30k yr BP. METHODS: The study was carried out by macroscopic and radiological analysis. RESULTS: The specimen has unusual morphology, with two vertebrae (C6-C7) connected in the ventrodorsal projection by osseous tissue, without a space or disruption between them. However, a separation is visible in the dorsoventral projection. Moreover, C7 shows a "wedge-shape" conformation. CONCLUSIONS: The lack of clear radiological and macroscopic evidence of degenerative processes and trauma suggests a congenital anomaly or pathology. The short height of the ventral margin of the block and evidence of a radiological 'waist' lead us to propose congenital block vertebra (CBV) as the most likely diagnosis. SIGNIFICANCE: The Cueva de Guantes specimen would be the first reported evidence of CBV in a Pleistocene cave bear (Ursus spelaeus). LIMITATIONS: All diagnosis of archaeological animal remains should be undertaken with caution, especially when based on partial remains, as in this case. Moreover, this specimen lacks the neural arches of C6 and C7, preventing evaluation of the vertebral foramina. SUGGESTIONS FOR FURTHER RESEARCH: Intensive review of cave bear skeletal collections is advised to find new cases and perform an epidemiological approach to the palaeopathology of cave bears.

Holocene history of a lake filling and vegetation dynamics of the Serra Sul dos Carajás, southeast Amazonia.

Down-core changes in sedimentary facies, elemental geochemistry, pollen, spore, δ13C, δ15N and radiocarbon records from a filled lake, named R4, of the Serra Sul dos Carajás were used to study the relationship between the paleomorphological and paleoecological processes and their significance for Holocene paleoclimatology of the southeast Amazonia. The sediment deposition of the R4 lake started around 9500 cal yr BP. Increase of detrital components from 9500 to 7000 cal yr BP suggests high weathering of surrounding catchment rocks and soils, and deposition into the lake basin under mudflows. At that time, montane savanna and forest formation were already established suggesting predominance of wet climate. However, from 7000 to 3000 cal yr BP, a decline of detrital input occurred. Also, forest formation and pteridophytes were declined, while palms and macrophytes were remained relatively stable, indicating that water levels of the lake is likely dropped allowing the development of plants adapted to subaerial condition under drier climate conditions. After 3000 cal yr BP, eutrophication and low accommodation space lead to high lake productivity and the final stage of the lake filling respectively, and forest formation may has acquired its current structure, which suggests return of wetter climate conditions.

Reduction spheroids preserve a uranium isotope record of the ancient deep continental biosphere.

Life on Earth extends to several kilometres below the land surface and seafloor. This deep biosphere is second only to plants in its total biomass, is metabolically active and diverse, and is likely to have played critical roles over geological time in the evolution of microbial diversity, diagenetic processes and biogeochemical cycles. However, these roles are obscured by a paucity of fossil and geochemical evidence. Here we apply the recently developed uranium-isotope proxy for biological uranium reduction to reduction spheroids in continental rocks (red beds). Although these common palaeo-redox features have previously been suggested to reflect deep bacterial activity, unequivocal evidence for biogenicity has been lacking. Our analyses reveal that the uranium present in reduction spheroids is isotopically heavy, which is most parsimoniously explained as a signal of ancient bacterial uranium reduction, revealing a compelling record of Earth's deep biosphere.

Contrasting patterns of prehistoric human diet and subsistence in northernmost Europe.

Current archaeological evidence indicates the transition from hunting-fishing-gathering to agriculture in Northern Europe was a gradual process. This transition was especially complex in the prehistoric North Fennoscandian landscape where the high latitude posed a challenge to both domestic animal breeding and cereal cultivation. The conditions varied, the coastal dwellers had access to rich marine resources and enjoyed a milder climate due to the Gulf Stream, while those living in the inland Boreal forest zone faced longer and colder winters and less diversity in animal and plant resources. Thus, the coastal area provided more favourable conditions for early agriculture compared to those found inland. Interestingly, a cultural differentiation between these areas is archaeologically visible from the late 2nd millennium BC onwards. This is most clearly seen in regionally distinct pottery styles, offering unique opportunities to probe diet and subsistence through the organic residues preserved in ceramic vessels. Herein, we integrate the lipid biomarker, compound-specific stable carbon isotopes (δ13C), and zooarchaeological evidence to reveal culturally distinct human diets and subsistence patterns. In northern Norway, some of the coastal people adopted dairying as part of their subsistence strategy, while the inhabitants of the interior, in common with northern Finland, continued their hunter-gatherer-fisher lifestyles.

Using Paleoecology to Inform Land Management as Climates Change: An Example from an Oak Savanna Ecosystem.

Oak savanna, a transitional ecosystem between open prairie and dense oak forest, was once widespread in Minnesota. Upon European settlement much of the oak savanna was destroyed. Recently, efforts to restore this ecosystem have increased and often include the reintroduction of fire. Though fire is known to serve an important role within oak savannas, there are currently few studies which address fire regimes on timescales longer than the last century. This research presents a paleoecological history of Sherburne National Wildlife Refuge (SNWR) in MN, USA, spanning the last ~8000 years. The objectives of this study were to use charcoal, pollen, and magnetic susceptibility of lake sediments collected from Johnson Slough (JS) within the refuge to evaluate the natural range of variability and disturbance history of the oak savanna within the refuge, assess the success of current restoration strategies, and add to the regional paleoecological history. The mid/late Holocene period of the JS record shows a period of high fire activity from ca. 6500 to 2600 cal year BP, with a shift from prairie to oak savanna occurring over this same period. A (possibly agricultural) disturbance to JS sediments affected the period from ca. 2600 cal year BP to 1963 AD, which includes the time of Euro-American settlement. However, the destruction and subsequent restoration of the oak savanna is evident in a pollen ratio of Quercus:Poaceae, indicating that current restoration efforts have been successful at restoring the oak savanna to within the natural range of variability seen just prior to destruction.

The earliest settlers of Mesoamerica date back to the late Pleistocene.

Preceramic human skeletal remains preserved in submerged caves near Tulum in the Mexican state of Quintana Roo, Mexico, reveal conflicting results regarding 14C dating. Here we use U-series techniques for dating a stalagmite overgrowing the pelvis of a human skeleton discovered in the submerged Chan Hol cave. The oldest closed system U/Th age comes from around 21 mm above the pelvis defining the terminus ante quem for the pelvis to 11311±370 y BP. However, the skeleton might be considerable older, probably as old as 13 ky BP as indicated by the speleothem stable isotope data. The Chan Hol individual confirms a late Pleistocene settling of Mesoamerica and represents one of the oldest human osteological remains in America.

A decorated raven bone from the Zaskalnaya VI (Kolosovskaya) Neanderthal site, Crimea.

We analyze a radius bone fragment of a raven (Corvus corax) from Zaskalnaya VI rock shelter, Crimea. The object bears seven notches and comes from an archaeological level attributed to a Micoquian industry dated to between 38 and 43 cal kyr BP. Our study aims to examine the degree of regularity and intentionality of this set of notches through their technological and morphometric analysis, complemented by comparative experimental work. Microscopic analysis of the notches indicate that they were produced by the to-and-fro movement of a lithic cutting edge and that two notches were added to fill in the gap left between previously cut notches, probably to increase the visual consistency of the pattern. Multivariate analysis of morphometric data recorded on the archaeological notches and sets of notches cut by nine modern experimenters on radii of domestic turkeys shows that the variations recorded on the Zaskalnaya set are comparable to experimental sets made with the aim of producing similar, parallel, equidistant notches. Identification of the Weber Fraction, the constant that accounts for error in human perception, for equidistant notches cut on bone rods and its application to the Zaskalnaya set of notches and thirty-six sets of notches incised on seventeen Upper Palaeolithic bone objects from seven sites indicate that the Zaskalnaya set falls within the range of variation of regularly spaced experimental and Upper Palaeolithic sets of notches. This suggests that even if the production of the notches may have had a utilitarian reason the notches were made with the goal of producing a visually consistent pattern. This object represents the first instance of a bird bone from a Neanderthal site bearing modifications that cannot be explained as the result of butchery activities and for which a symbolic argument can be built on direct rather than circumstantial evidence.

Archaeological occurrences and historical review of the human amoeba, Entamoeba histolytica, over the past 6000years.

Understanding parasite history and the evolution of host/parasite relationships is one of the most important aspects of paleoparasitology. Within the framework of this research topic, this paper focuses on the human pathogenic amoeba, Entamoeba histolytica. The compilation of all the available archaeological data concerning this parasite leads to a first glimpse of the history of this parasite of current medical importance. Paleoparasitological investigation into this parasite uses immunological techniques and shows that the modern strain of E. histolytica has been present in Western Europe since at least the Neolithic period (3700yearsBCE), and could have originated in the Old World. The appearance of the modern amoeba strain in the pre-Columbian Americas and the Middle East around the 12th century CE gives rise to hypotheses as to how human migrations (Atlantic or Pacific routes) contributed to the diffusion of this pathogen, resulting in its current distribution. This compilation proves that parasites are valuable proxies for studying past human and animal migrations, and should be given more consideration in the future.

How have studies of ancient DNA from sediments contributed to the reconstruction of Quaternary floras?

499 I. 499 II. 500 III. 500 IV. 500 V. 500 VI. 501 VII. 502 VIII. 504 504 References 505 SUMMARY: Ancient DNA (aDNA) from lake sediments, peats, permafrost soils, preserved megafaunal gut contents and coprolites has been used to reconstruct late-Quaternary floras. aDNA is either used alone for floristic reconstruction or compared with pollen and/or macrofossil results. In comparative studies, aDNA may complement pollen and macrofossil analyses by increasing the number of taxa found. We discuss the relative contributions of each fossil group to taxon richness and the number of unique taxa found, and situations in which aDNA has refined pollen identifications. Pressing problems in aDNA studies are contamination and ignorance about taphonomy (transportation, incorporation, and preservation in sediments). Progress requires that these problems are reduced to allow aDNA to reach its full potential contribution to reconstructions of Quaternary floras.

Archaeological bone lipids as palaeodietary markers.

RATIONALE: Stable isotope analysis of archaeological and fossil bone samples can provide important insights into past environments, ecologies and diets. Previous studies have focused on stable carbon and nitrogen isotopes in bone collagen, or carbon isotopes in bone mineral (bioapatite). Carbon isotope analysis of lipids from archaeological bone has received much less attention, partly due to the lack of suitable methodologies allowing sufficient recovery of compounds for structural and isotopic characterisation. Here we show that lipids can be easily and reliably recovered from archaeological bone using a modified protocol, and that these provide complementary dietary information to other bone components. METHODS: Human and animal bones were obtained from a variety of archaeological contexts. Lipids were sequentially extracted using solvent extraction (dichloromethane/methanol), followed by acidified methanol extraction (methanol/H2SO4). The lipids were then analysed by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). RESULTS: Appreciable amounts of endogenous lipid were recovered from archaeological bone. Importantly, a comparison between compound-specific and bulk collagen isotopic data shows that archaeological bone lipids reflect dietary input and can be used to distinguish between marine and terrestrial consumers, as well as between C3 and C4 plant consumers. Furthermore, the presence of essential fatty acids directly incorporated from diet to bone may provide additional palaeodietary information. CONCLUSIONS: Our findings suggest that archaeological bone lipids are a hitherto untapped resource of dietary information that offer additional insights to those gained from other isotopic analyses of bone.

Uranium isotopes fingerprint biotic reduction.

Knowledge of paleo-redox conditions in the Earth's history provides a window into events that shaped the evolution of life on our planet. The role of microbial activity in paleo-redox processes remains unexplored due to the inability to discriminate biotic from abiotic redox transformations in the rock record. The ability to deconvolute these two processes would provide a means to identify environmental niches in which microbial activity was prevalent at a specific time in paleo-history and to correlate specific biogeochemical events with the corresponding microbial metabolism. Here, we demonstrate that the isotopic signature associated with microbial reduction of hexavalent uranium (U), i.e., the accumulation of the heavy isotope in the U(IV) phase, is readily distinguishable from that generated by abiotic uranium reduction in laboratory experiments. Thus, isotope signatures preserved in the geologic record through the reductive precipitation of uranium may provide the sought-after tool to probe for biotic processes. Because uranium is a common element in the Earth's crust and a wide variety of metabolic groups of microorganisms catalyze the biological reduction of U(VI), this tool is applicable to a multiplicity of geological epochs and terrestrial environments. The findings of this study indicate that biological activity contributed to the formation of many authigenic U deposits, including sandstone U deposits of various ages, as well as modern, Cretaceous, and Archean black shales. Additionally, engineered bioremediation activities also exhibit a biotic signature, suggesting that, although multiple pathways may be involved in the reduction, direct enzymatic reduction contributes substantially to the immobilization of uranium.

The Neanderthal in the karst: First dating, morphometric, and paleogenetic data on the fossil skeleton from Altamura (Italy).

In 1993, a fossil hominin skeleton was discovered in the karst caves of Lamalunga, near Altamura, in southern Italy. Despite the fact that this specimen represents one of the most extraordinary hominin specimens ever found in Europe, for the last two decades our knowledge of it has been based purely on the documented on-site observations. Recently, the retrieval from the cave of a fragment of bone (part of the right scapula) allowed the first dating of the individual, the quantitative analysis of a diagnostic morphological feature, and a preliminary paleogenetic characterization of this hominin skeleton from Altamura. Overall, the results concur in indicating that it belongs to the hypodigm of Homo neanderthalensis, with some phenetic peculiarities that appear consistent with a chronology ranging from 172 ± 15 ka to 130.1 ± 1.9 ka. Thus, the skeleton from Altamura represents the most ancient Neanderthal from which endogenous DNA has ever been extracted.

Unravelling the complexity of domestication: a case study using morphometrics and ancient DNA analyses of archaeological pigs from Romania.

Current evidence suggests that pigs were first domesticated in Eastern Anatolia during the ninth millennium cal BC before dispersing into Europe with Early Neolithic farmers from the beginning of the seventh millennium. Recent ancient DNA (aDNA) research also indicates the incorporation of European wild boar into domestic stock during the Neolithization process. In order to establish the timing of the arrival of domestic pigs into Europe, and to test hypotheses regarding the role European wild boar played in the domestication process, we combined a geometric morphometric analysis (allowing us to combine tooth size and shape) of 449 Romanian ancient teeth with aDNA analysis. Our results firstly substantiate claims that the first domestic pigs in Romania possessed the same mtDNA signatures found in Neolithic pigs in west and central Anatolia. Second, we identified a significant proportion of individuals with large molars whose tooth shape matched that of archaeological (likely) domestic pigs. These large 'domestic shape' specimens were present from the outset of the Romanian Neolithic (6100-5500 cal BC) through to later prehistory, suggesting a long history of admixture between introduced domestic pigs and local wild boar. Finally, we confirmed a turnover in mitochondrial lineages found in domestic pigs, possibly coincident with human migration into Anatolia and the Levant that occurred in later prehistory.

Reassessing the biogenicity of Earth's oldest trace fossil with implications for biosignatures in the search for early life.

Microtextures in metavolcanic pillow lavas from the Barberton greenstone belt of South Africa have been argued to represent Earth's oldest trace fossil, preserving evidence for microbial life in the Paleoarchean subseafloor. In this study we present new in situ U-Pb age, metamorphic, and morphological data on these titanite microtextures from fresh drill cores intercepting the type locality. A filamentous microtexture representing a candidate biosignature yields a U-Pb titanite age of 2.819 ± 0.2 Ga. In the same drill core hornfelsic-textured titanite discovered adjacent to a local mafic sill records an indistinguishable U-Pb age of 2.913 ± 0.31 Ga, overlapping with the estimated age of intrusion. Quantitative microscale compositional mapping, combined with chlorite thermodynamic modeling, reveals that the titanite filaments are best developed in relatively low-temperature microdomains of the chlorite matrix. We find that the microtextures exhibit a morphological continuum that bears no similarity to candidate biotextures found in the modern oceanic crust. These new findings indicate that the titanite formed during late Archean ca. 2.9 Ga thermal contact metamorphism and not in an early ca. 3.45 Ga subseafloor environment. We therefore question the syngenicity and biogenicity of these purported trace fossils. It is argued herein that the titanite microtextures are more likely abiotic porphyroblasts of thermal contact metamorphic origin that record late-stage retrograde cooling in the pillow lava country rock. A full characterization of low-temperature metamorphic events and alternative biosignatures in greenstone belt pillow lavas is thus required before candidate traces of life can be confirmed in Archean subseafloor environments.