Recovering ancient parasites from Andean herbivores: test of the Mini-FLOTAC technique in archaeological samples.

The optimization of techniques for recovering parasitic remains is key in paleoparasitology. The Mini-FLOTAC technique (MF) is based on passive flotation and is used for diagnosis of parasites and was never tested on ancient samples. Our objective was to assess the effectiveness of MF in paleoparasitology, aiming at improving the techniques for the recovery of parasitic remains in order to upgrade the interpretative potential of the paleoparasitological evidence. Three techniques were tested: MF, spontaneous sedimentation (SS), and centrifugation-sucrose flotation (CF) testing camelid and goat coprolites. Statistical tests were performed with the R software. Our result displayed that, in the case of SAC samples, MF recovered less number of parasitic species than SS, but obtained a greater number of positive samples for protozoa. For goat samples, MF recovered a higher number of positive samples and parasitic species than SS, added that it was the technique that recovered a greater number of parasite structures. Therefore, results vary according to the zoological origin of the samples and the parasitic species recorded. We suggest starting using MF on ancient samples as a complementary method to those traditionally used in paleoparasitology. It is important to highlight that MF was a simple and faster way. The incorporation of reliable quantitative techniques opens the door to a new way of analyzing archaeological remains, deepening the study of the parasite-host relationships and its evolution through time with an epidemiological approach. Although further studies are needed, our results suggest the complementarity of these techniques in future paleoparasitological studies.

Trichurid nematodes from South American Camelid: an approach to native assemblages through the parasitology of archaeological sites.

To gain insights into the trichurid diversity in pre-Hispanic remains of South American camelids (SACs), as well as into modern SAC populations, a bibliographic search was carried out to find parasitological studies. Results showed that a total of 17 studies were performed at archaeological and paleontological sites, in which a total of 213 samples were examined. The sample set was used as the unit of analysis, and 83% of 52 pre-Hispanic sample sets were positive for parasites. Parasitological studies confirmed the pre-Hispanic presence of diverse trichurids: two Trichuris species and capillariid eggs with morphological differences that allowed them to be assigned to three different species. Currently, the richness of the native trichurid assemblages in SACs has been extended by the inclusion of two species (Trichuris sp. 2 and Capillariinae gen. sp. 1). Modern T. tenuis Chandler, 1930, considered SAC-specific, would have invaded SAC populations during the European colonization. The estimate of regional and temporal trichurid prevalence and paleoecological information allowed us to propose a possible origin for some of them. Although the present analysis is exploratory, it should stimulate future studies about the biogeographic history of SAC helminths, provide relevant information to improve descriptions of the Holocene ecology, and identify threatened helminths.

Assessing parasite epidemiology in the epidemiological importance of Patagonian Late Holocene rockshelters using carnivore coprolites in the past: new data from Piedra Parada, Argentina.

The aim of this research was to analyse the variability of parasite assemblages on a small spatial scale, by examining carnivore coprolites from the archaeological site Campo Moncada 2 (CM2), Piedra Parada area, Chubut province, Argentina, and comparing the results with those previously obtained from the archaeological site Campo Cerda 1 (CCe1), located in the same area. Six carnivore coprolites from CM2 were analysed: 4 obtained in sub-level 2a and 2 obtained in sub-level 2a/b. Two radiocarbon dates associated with the coprolites placed the samples chronologically between 780 ± 80 and 860 ± 80 years before present. The rehydrated sediments were sieved and then allowed to sediment spontaneously. The sediment was used for parasitological examination under light microscopy. Conservative estimation of total parasite richness resulted in 21 parasitic taxa. The taxa with the highest fecal prevalence (>50%) corresponded to parasites prevalent in modern carnivores (Alaria sp., Toxocara cf. canis, Toxascaris sp., Eucoleus cf. aerophila, Trichuris sp. and Ancylostomatidae gen. sp.). Assuming that the fox coprolites are contemporaneous, the total fecal parasite richness estimated for CM2 and previously for CCe1 was similar. The high total parasite richness found suggests a network of host­parasite relationships that could include regional hunter-gatherers. The results obtained in carnivore coprolites allow us to infer a very diverse biological community in Piedra Parada area, so the regional caves and rockshelters could have a proportional epidemiological importance as parasite exchange nodes.

Evidence of Toxocara Eggs in Pachycrocuta brevirostris (Gervais, 1850) Coprolites from the Pleistocene of Taurida Cave (Crimea).

Coprolites of the hyena Pachycrocuta brevirostris from the Lower Pleistocene (Upper Villafranchian) of Taurida Cave (Crimea) were studied. One of the three hyena coprolites contained helminth eggs. These eggs were assigned to Toxocara sp. based on their size and morphology. Toxocariasis was evidently a very common infestation among extinct hyena species. The find of toxocara in P. brevirostris coprolite from the Taurida Cave is the earliest evidence of roundworm infestation in P. brevirostris.

The erroneous chondrichthyan egg case assignments from the Devonian: implications for the knowledge on the evolution of the reproductive strategy within chondrichthyans.

Spiraxis interstrialis, and its junior synonym Fayolia mourloni, an uppermost Famennian (Upper Devonian) fossil first described as algae and subsequently interpreted as the oldest known chondrichthyan egg case, is reinvestigated based on the discovery of several additional specimens in Belgian collections. New data, in particular from micro-CT imaging, allow to refute S. interstrialis, and by extension also Spiraxis major (the type species of Spiraxis Newberry, non Adams) and Spiraxis randalli from the Famennian of New York and Pennsylvania, as chondrichthyan egg cases. Alternative interpretations of these enigmatic helicoidal fossils are discussed. The first occurrence of oviparity in the fossil record of chondrichthyans is thus not as old as previously thought and is close to the first occurrence of viviparity in this group, both being recognised now in the Mississippian. The question of which of both conditions is plesiomorphic within chondrichthyans, and more widely within vertebrates, is discussed. Also, the presence of the genus Spiraxis in both the USA (east coast) and Belgium reinforces the strong faunal resemblance already observed in both palaeogeographical areas. It suggests important faunal exchanges between these regions of the Euramerica landmass during the Famennian.

Coprolites reveal ecological interactions lost with the extinction of New Zealand birds.

Over the past 50,000 y, biotic extinctions and declines have left a legacy of vacant niches and broken ecological interactions across global terrestrial ecosystems. Reconstructing the natural, unmodified ecosystems that preceded these events relies on high-resolution analyses of paleoecological deposits. Coprolites are a source of uniquely detailed information about trophic interactions and the behaviors, gut parasite communities, and microbiotas of prehistoric animal species. Such insights are critical for understanding the legacy effects of extinctions on ecosystems, and can help guide contemporary conservation and ecosystem restoration efforts. Here we use high-throughput sequencing (HTS) of ancient eukaryotic DNA from coprolites to reconstruct aspects of the biology and ecology of four species of extinct moa and the critically endangered kakapo parrot from New Zealand (NZ). Importantly, we provide evidence that moa and prehistoric kakapo consumed ectomycorrhizal fungi, suggesting these birds played a role in dispersing fungi that are key to NZ's natural forest ecosystems. We also provide the first DNA-based evidence that moa frequently supplemented their broad diets with ferns and mosses. Finally, we also find parasite taxa that provide insight into moa behavior, and present data supporting the hypothesis of coextinction between moa and several parasite species. Our study demonstrates that HTS sequencing of coprolites provides a powerful tool for resolving key aspects of ancient ecosystems and may rapidly provide information not obtainable by conventional paleoecological techniques, such as fossil analyses.

Parasitological study of mountain viscacha fecal pellets from patagonia over the last 1200 years ('Cueva Peligro', Chubut province, Argentina).

The aim of the present study was to examine the parasite fauna present in mountain viscacha Lagidium viscacia (Caviomorpha, Chinchillidae) fecal pellets collected from 'Cueva Peligro', a cave located in Chubut province, Patagonia, Argentina, throughout the last 1200 years. A total of 84 samples were examined for parasites. Each pellet was whole processed: rehydrated, homogenized, sediment and examined using light microscopy. The samples and eggs of parasites present were described, measured and photographed. Thirty-eight samples tested positive for the nematodes Heteroxynema (Cavioxyura) viscaciae Sutton & Hugot, 1989, Helminthoxys effilatus Schuurmans-Stekhoven, 1951 (Oxyurida: Oxyuridae), Trichuris sp. Roederer, 1761 (Trichinellida: Trichuridae) and one anoplocephalid species (Cestoda: Anoplocephalidae). This is the first time that H. effilatus is reported from ancient times. Significant differences of parasite occurrences through this temporal period were recorded. Parasitic life cycles and their presence along the studied period are so discussed.

Ancient parasites from endemic deer from "CUEVA PARQUE DIANA" archeological site, Patagonia, Argentina.

The narrow Andean-Patagonian temperate rainforest strip in the west of southern South America is inhabited by two endemic species of cervids, the southern pudu (Pudu puda) and the huemul (Hippocamelus bisulcus), both cataloged as near threatened and threatened species, respectively. One of the possible causes of their declined number is the susceptibility to livestock diseases. Significant zooarchaeological records of both deer have been found throughout the Holocene from Patagonia. The present contribution reports the first paleoparasitological results obtained from coprolites of endemic deer from the archeological site "Cueva Parque Diana," Neuquén Province, Argentina, and discusses the possible diseases found in ancient times. Thirty-four coprolites were fully processed, rehydrated, homogenized, sieved, subjected to spontaneous sedimentation, and examined by light microscopy. Thirty samples contained parasite remains. The presence of diverse parasitic diseases such as trematodioses, metastrongylosis, trichuriosis, strongylida gastroenteritis, dioctophymosis, and coccidiosis which could cause diseases in deer previous to the arrival of European livestock and the presence of zoonotic diseases in the hunters-gatherers and fishermen are discussed.

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.

Scale insect larvae preserved in vertebrate coprolites (Le Quesnoy, France, Lower Eocene): paleoecological insights.

Coprolites of terrestrial vertebrates from the Sparnacian Le Quesnoy locality (Ypresian, Eocene, MP7, 53 Ma; Oise, France) were examined for possible parasitic helminth eggs. The extraction of the coprolite components was performed by a weak acetolyse and a slide mounting in glycerin. This long examination did not reveal paleoparasite remains, which may be explained through several arguments. However, some pollen grains, some enigmatic components, and two well-preserved first-instar cochineal nymphs (Hemiptera: Sternorrhyncha: Coccoidea) were evidenced in coprolites. Identified as Coccidae, these larvae are the earliest stage of the scale insect development ever reported as fossil, revealing the specific environment of preservation that fossilized scats may provide. These observations, combined to the coprolites morphotype, enable to ascribe the fossil scats producer to a small herbivorous mammal present in the deposit (early perissodactyls or Plesiadapidae). Regarding the ecology of extant representatives of Coccidae, this mammal was a likely foliage consumer, and the abundant Juglandaceae and/or Tiliaceae from Le Quesnoy might have lived parasitized by scale insects. These Early Eocene parasites had an already well-established dissemination strategy, with prevalent minute first-instar larvae. The herein performed extraction technique appears well-suited for the study of carbonate coprolites and could certainly be useful for evidencing other kind of microorganisms (including internal parasites).

Protozoan parasites of birds from the Tremembé formation (Oligocene of the Taubaté Basin), São Paulo, Brazil.

OBJECTIVE: To analyze the presence of protozoan parasites in bird coprolites from the Tremembé Formation (Oligocene of the Taubaté Basin). MATERIALS: Twenty avian coprolites embedded in pyrobituminous shale matrices. METHODS: Samples were rehydrated and subjected to spontaneous sedimentation. RESULTS: Paleoparasitological analyses revealed oocysts compatible with the Eimeriidae family (Apicomplexa) and one single Archamoebae (Amoebozoa) cyst. CONCLUSIONS: The present work increases the amount of information about the spread of infections throughout the Cenozoic Era and reveals that the Brazilian paleoavifauna played an important role in the Apicomplexa and Amoebozoa life cycles. SIGNIFICANCE: This is the first record of protozoans in avian coprolites from the Oligocene of Brazil. These findings can help in the interpretation of phylogenies of coccidian parasites of modern birds, as certain taxonomic characters observed in the Oligocene Protozoa characterize monophyletic groups in current molecular phylogenetic analyses. LIMITATIONS: None of the oocysts were sporulated; therefore, it is not possible to identify the morphotypes to genus or species. SUGGESTIONS FOR FURTHER RESEARCH: Our results create new perspectives related to biogeographic studies of the parasitic groups described and may improve the understanding of the temporal amplitude of parasitic evolutionary relationships between Protozoans and birds.

First record of well-preserved canid coprolites from Eurasia: New insights into the unique ecological niche of Yuanmou Basin.

This study reconstructs the Early Pleistocene paleoenvironment of the Yuanmou Basin through coproecology of the third member of the Yuanmou Formation. We examined 38 exceptionally well-preserved coprolites from a new fossil locality, and attributed the putative defecating agent to the hypercarnivorous diet canid, Sinocuon yuanmouensis through geochemical and quantitative analyses. A new ichnogenus and ichnospecies, Cuocopros yuanmouensis igen. et. isp. nov., was established based on distinctive characteristics. Multi-disciplinary analysis, including sediment palynology and lithostratigraphy, helped primarily reconstruct a significant climatic event during the early Pleistocene, coinciding with the emergence of Yuanmou Man during the fourth member of the Yuanmou Formation's deposition. The findings provide insights into coexistence between canids, hyaenas, hominoids, and other fauna, revealing a rich paleoecosystem and food chain in the region's history. This study contributes to understanding the complex ecological dynamics during this period in the Yuanmou Basin.

Fungal-induced fossil biomineralization.

Exceptional preservation of fossils has often been attributed to the actions of bacteria that aid in the preservation of soft tissues that normally decay rapidly. However, it is well known that fungi play a major role in organic matter decomposition, biogeochemical cycling of elements, and metal-mineral transformations in modern ecosystems. Although the fungal fossil record can be traced back over a billion years, there are only a few recorded examples of fungal roles in fossilization. In this research, we have carried out a detailed geobiological investigation on early Pleistocene hyena coprolites (fossilized dung) in an attempt to ascertain possible fungal involvement in their formation. Using an advanced microscopic and mineralogical approach, we found that numerous hydroxyapatite nanofibers (25-34 nm on average), interwoven to form spheroidal structures, constituted the matrix of the coprolites in addition to food remains. These structures were found to be extremely similar in texture and mineral composition to biominerals produced during laboratory culture of a common saprophytic and geoactive fungus, Aspergillus niger, in the presence of a solid source of calcium (Ca) and phosphorus (P). This observation, and our other data obtained, strongly suggests that fungal metabolism can provide a mechanism that can result in fossil biomineralization, and we hypothesize, therefore, that this may have contributed to the formation of well-preserved fossils (Lagerstätten) in the geological record. The characteristic polycrystalline nanofibers may also have served as a potential biosignature for fungal life in early Earth and extraterrestrial environments.

Fossil Biomarkers and Biosignatures Preserved in Coprolites Reveal Carnivorous Diets in the Carboniferous Mazon Creek Ecosystem.

The reconstruction of ancient trophic networks is pivotal to our understanding of ecosystem function and change through time. However, inferring dietary relationships in enigmatic ecosystems dominated by organisms without modern analogues, such as the Carboniferous Mazon Creek fauna, has previously been considered challenging: preserved coprolites often do not retain sufficient morphology to identify the dietary composition. Here, we analysed n = 3 Mazon Creek coprolites in concretions for dietary signals in preserved biomarkers, stable carbon isotope data, and macromolecular composition. Cholesteroids, metazoan markers of cholesterol, show an increased abundance in the sampled coprolites (86 to 99% of the total steranes) compared to the surrounding sediment, indicating an endogenous nature of preserved organics. Presence of unaltered 5α-cholestan-3ß-ol and coprostanol underline the exceptional molecular preservation of the coprolites, and reveal a carnivorous diet for the coprolite producer. Statistical analyses of in situ Raman spectra targeting coprolite carbonaceous remains support a metazoan affinity of the digested fossil remains, and suggest a high trophic level for the coprolite producer. These currently oldest, intact dietary stanols, combined with exquisitely preserved macromolecular biosignatures in Carboniferous fossils offer a novel source of trophic information. Molecular and biosignature preservation is facilitated by rapid sedimentary encapsulation of the coprolites within days to months after egestion.

Analysis of microbiome diversity in coprolites from Caral, Peru.

We analyzed human coprolites from the Sacred City of Caral, the oldest civilization in America (3000- and 1800-years BC). Our objective was to know the microbial diversity of the Caral Civilization through the use of a mobile ancient laboratory. DNA extraction conducted in a mobile laboratory placed near the collection site to reduce exposure of samples to contaminants and favor a rapid molecular processing. Using 16S rRNA and ITS 1 amplicon sequencing, we have elaborated the first list of the microbiomes of Caral, based on the bacterial and fungal community fingerprints detected in the coprolites recovered in six sectors of that ancient urban center. Among the most abundant sequences were those associated with Firmicutes for bacteria, Ascomycota and Basidiomycota for fungi. Bacillus was the most abundant bacterial genera in all samples analyzed, compromising up to 24.81% of the total bacterial abundance; while Aspergillus (11.43%) was the most abundant genera among fungal communities.

Comparative actualistic study hints at origins of alleged Miocene coprolites of Poland.

Excrement-shaped ferruginous masses have been recovered from the Miocene of Turów mine in south-western Poland. These siderite masses have been the subject of much controversy, having been interpreted either as being coprolites, cololithes or pseudofossils created by mechanical deformation of plastic sediment. Here we present the results of mineralogical, geochemical, petrographic and microtomographical analyses. Our data indicate that these masses consist of siderite and iron oxide rather than phosphate, and rarely contain recognizable food residues, which may suggest abiotic origins of these structures. On the other hand, evidence in support of a fecal origin include: (i) the presence of two distinct morphotypes differing in size and shape, (ii) the presence of rare hair-like structures or coalified inclusions and (iii) the presence of rare fine striations on the surface. Importantly, comparative actualistic study of recent vertebrate feces shows overall resemblance of the first morphotype (sausage-shaped with rare coalified debris) to excrements of testudinoid turtles (Testudinoidea), whose shell fragment was found in the investigated locality. The second morphotype (rounded to oval-shaped with hair-like structures), in turn, is similar to the feces of some snakes (Serpentes), the remains of which were noted in the Miocene of the neighborhood areas. Other potential producers (such as lizards and crocodiles) and even abiotic origins cannot be fully excluded but are less likely.

A mobile lab for ancient DNA extraction in Perug.

We report the use of a mobile laboratory set up to extract ancient DNA (aDNA) from 34 human coprolites (fossilized faeces) samples. Our approach enabled the rapid genetic characterization of 5,000 years old archeological samples. It is useful for the on-site screening of museums and freshly excavated samples for DNA. This approach is accessible to other investigators as the mobile laboratory was set up using commercially available instruments.

Exceptionally preserved beetles in a Triassic coprolite of putative dinosauriform origin.

The Triassic was a crucial period for the early evolution and diversification of insects, including Coleoptera1-3-the most diverse order of organisms on Earth. The study of Triassic beetles, however, relies almost exclusively on flattened fossils with limited character preservation. Using synchrotron microtomography, we investigated a fragmentary Upper Triassic coprolite, which contains a rich record of 3D-preserved minute beetle remains of Triamyxa coprolithica gen. et sp. nov. Some specimens are nearly complete, preserving delicate structures of the legs and antennae. Most of them are congruent morphologically, implying that they are conspecific. Phylogenetic analyses suggest that T. coprolithica is a member of Myxophaga, a small suborder of beetles with a sparse fossil record, and that it represents the only member of the extinct family Triamyxidae fam. nov. Our findings highlight that coprolites can contain insect remains, which are almost as well preserved as in amber. They are thus an important source of information for exploring insect evolution before the Cretaceous-Neogene "amber time window." Treated as food residues, insect remains preserved in coprolites also have important implications for the paleoecology of insectivores, in this case, likely the dinosauriform Silesaurus opolensis.

Functional diversity of microbial ecologies estimated from ancient human coprolites and dental calculus.

Human microbiome studies are increasingly incorporating macroecological approaches, such as community assembly, network analysis and functional redundancy to more fully characterize the microbiome. Such analyses have not been applied to ancient human microbiomes, preventing insights into human microbiome evolution. We address this issue by analysing published ancient microbiome datasets: coprolites from Rio Zape (n = 7; 700 CE Mexico) and historic dental calculus (n = 44; 1770-1855 CE, UK), as well as two novel dental calculus datasets: Maya (n = 7; 170 BCE-885 CE, Belize) and Nuragic Sardinians (n = 11; 1400-850 BCE, Italy). Periodontitis-associated bacteria (Treponema denticola, Fusobacterium nucleatum and Eubacterium saphenum) were identified as keystone taxa in the dental calculus datasets. Coprolite keystone taxa included known short-chain fatty acid producers (Eubacterium biforme, Phascolarctobacterium succinatutens) and potentially disease-associated bacteria (Escherichia, Brachyspira). Overlap in ecological profiles between ancient and modern microbiomes was indicated by similarity in functional response diversity profiles between contemporary hunter-gatherers and ancient coprolites, as well as parallels between ancient Maya, historic UK, and modern Spanish dental calculus; however, the ancient Nuragic dental calculus shows a distinct ecological structure. We detected key ecological signatures from ancient microbiome data, paving the way to expand understanding of human microbiome evolution. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

Parasites in micromammal fecal pellets throughout the Late Holocene ("Cueva Peligro" paleontological site, Patagonia, Argentina).

Parasite remains in micromammal fecal pellets collected from the paleontological site "Cueva Peligro" (CP 43°40'18"S, 66°24'52"W), Chubut Province, Argentina, were examined. The samples were obtained from two grids, dated between 1220 ± 7014C yr B.P. to modern dates. Fecal pellets were whole processed, rehydrated, homogenized, and examined via light microscopy. Eggs of parasites found were measured and photographed. Fecal pellets belong to one or more insectivore to omnivore unidentified micromammal species, possibly sigmodontine rodents. A high number of helminthes species was recorded (11 species), eight nematodes, two anoplocephalid cestodes and one acanthocephalan species. The sigmodontine-parasite relationship varied throughout the studied period and between studied grids. This is the first time that Gongylonema sp. and Syphacia sp. are reported from ancient times from Patagonia. The obtained results contribute to the knowledge of parasite assemblages associated to native South American sigmodontine rodents and the zoonoses present in the area throughout the lasts 1200 years.