Extraction Of Microfossils Research Articles

Corrigendum: Sequential biomolecular, macrofossil, and microfossil extraction from coprolites for reconstructing past behavior and environments

Corrigendum: Sequential biomolecular, macrofossil, and microfossil extraction from coprolites for reconstructing past behavior and environments

Sequential biomolecular, macrofossil, and microfossil extraction from coprolites for reconstructing past behavior and environments

The study of macroscopic, microscopic, and biomolecular remains recovered from coprolites can provide a wide range of information on past human and animal behavior and environments. In earlier studies, research tended to focus on one or two proxies, but multiproxy approaches combining data from all remains within coprolites are becoming more common. Multiproxy analyses have demonstrated value for strengthening our understanding of the past and reducing equifinality. Here we present a sequential biomolecular, macrofossil, and microfossil extraction protocol that separates all different coprolite components and is intended as a best-practice guideline for coprolite analysis. We demonstrate the effectiveness of this method by applying it to an assemblage of coprolites from the Paisley Caves, Oregon, USA. By combining a wide range of proxies, this study provides important information on the taxonomy and behavior of organisms in the past as well as the paleoecological context of behavior.

Inferring the age and environmental characteristics of fossil sites using citizen science.

Not all fossil sites preserve microfossils that can be extracted using acid digestion, which may leave knowledge gaps regarding a site's age or environmental characteristics. Here we report on a citizen science approach that was developed to identify microfossils in situ on the surface of sedimentary rocks. Samples were collected from McGraths Flat, a recently discovered Miocene rainforest lake deposit located in central New South Wales, Australia. Composed entirely of iron-oxyhydroxide, McGraths Flat rocks cannot be processed using typical microfossil extraction protocols e.g., acid digestion. Instead, scanning electron microscopy (SEM) was used to automatically acquire 25,200 high-resolution images from the surface of three McGraths Flat samples, covering a total area of 1.85 cm2. The images were published on the citizen science portal DigiVol, through which 271 citizen scientists helped to identify 300 pollen and spores. The microfossil information gained in this study is biostratigraphically relevant and can be used to constrain the environmental characteristics of McGraths Flat. Our findings suggest that automated image acquisition coupled with an evaluation by citizen scientists is an effective method of determining the age and environmental characteristics of fossiliferous rocks that cannot be investigated using traditional methods such as acid digestion.

Microfossils in resin from the middle Eocene Buchanan Lake Formation, Napartulik, Axel Heiberg Island, Nunavut, Canada

During the warm middle Eocene (ca 45 Ma), the Napartulik area (also called ‘the Geodetic Hills’), Axel Heiberg Island, northern Canada (Nunavut), was vegetated with mixed broad-leaved deciduous angiosperm and evergreen conifer forests over extensive floodplain and forested wetland habitats. Massive organic rich sedimentary successions and encapsulated in-situ tree trunks suggest these forests were drowned by frequent flooding events. The sedimentary layers contain sub-fossil amber that was produced by representatives of the Pinaceae such as Pseudolarix. The amber offered an opportunity to investigate aerial plankton and thus the chance to discover microfossils, which could provide evidence of biotic interactions associated with, or the cause of, the forest die-offs. Fifty-four amber samples were subjected to a solvent treatment for microfossil extraction followed by light microscopy, resulting in the discovery of several hundred microfossils. Unexpectedly, one-quarter of the microfossils were diatoms, which may predominantly have lived on the tree bark. Fungal spores were rare, and the pollen grains found corroborated earlier findings in litter or coal. The records of fungal spores and arthropod remains were insignificant and could, therefore, not substantially have been contributing to the forest die-offs. More resin analyses from the complete sedimentary profile would be needed to get a clearer picture of putative forest pests and prevailing environmental conditions

Investigating Biases Associated With Dietary Starch Incorporation and Retention With an Oral Biofilm Model

Dental calculus has proven to contain a wealth of information on the dietary habits of past populations. These insights have, to a large extent, been obtained by the extraction and identification of starch granules contained within the mineralised dental plaque from a wide range of regions and time periods. The scope of previous studies have been limited to microfossil extraction and identification to reconstruct dietary preferences from the archaeological record, and few studies have attempted to address the biases of starch retention in dental calculus. Those that have considered this problem have been limited to in vivo studies on modern humans and non-human primates. Here, we present a multispecies oral biofilm model, which allows experimental research on starch incorporation and retention to be conducted on in vitro dental calculus in a controlled laboratory setting. The biofilms were exposed to treatment solutions with known quantities of dietary starches (wheat and potato) during the 25 days growth period. After this, the starch granules were extracted from the mature biofilm (by dissolution in EDTA), and counted. We show that the granule counts extracted from the model dental calculus represented a low proportion (ranging from 0.06% to 0.16%) of the total number of granules exposed to the biofilms throughout the experiment. Additionally, we found that the ratios of granule sizes from the extracted starch granules differed from the original treatment solutions, with large granules (>20 μm) consistently being under-represented. We also found a positive correlation between the absolute granule counts and dry-weight of the biofilm (r = 0.659, 90%CI[0.463, 0.794]), meaning the absolute quantity of starch granules will increase as the size of the calculus deposit increases. A similar, but weaker correlation was found between the concentration (count per mg) of granules and dry-weight (r = 0.3, 90%CI[0.0618, 0.506]). Our results complement and reinforce previous in vivo studies suggesting that dental calculus presents a very small, and partly biased picture of the original dietary intake of starches, with an over-representation of plants producing granules smaller than 20 μm in size. The experimental model presented here is well-suited to address the need for further validation of methods and biases associated with dietary research on dental calculus.

Methodological Development of a Combined Preparation for Micropaleontological and Sedimentological Studies of Samples From the Proterozoic Record

The recovery of microfossils from Proterozoic rocks is commonly challenging because of metamorphism. In this study, an application of different methods usually applied on Phanerozoic rocks to test efficiency on recovering microfossil from Proterozoic units is presented. Chemical, physical, and biological factors can influence the recovery of microfossils, thereby becoming a barrier for biostratigraphic and paleoecological studies. Furthermore, low-cost projects with a reduced amount of sample collected, such as drill core sampling, need to optimize the preparation time and sample needed for different analyses. To overcome this challenge, the classical procedure of mineralized microfossil preparation, the palynological technique, and the study of clay mineralogy with the analyses of diagenetic alteration and the search for possible microfossils in thin sections were combined. Three Proterozoic lithostratigraphic units were selected to develop an integrated procedure for preparing samples for micropaleontologic and sedimentologic studies: the Paranoá Group, Mesoproterozoic, and the Bambuí Group, Ediacarian-Cambrian, Brazil, and Nama Group, Ediacaran-Cambrian, Namibia. Recovering individual microfossils from the Paranoá and Bambuí groups has been a challenge for paleontologists. Therefore, most micropaleontological studies have been done as a part of microbiofacies analyses in thin sections. All sediment fractions were studied in trial for the examination (and picking) of mineralized microfossils, even the finest ones. The microfossil picking was conducted using a stereomicroscope. Three species were recovered following this procedure: Vetronostocale aff V. amoenumSchopf and Blacic, 1971, Myxococcoides sp., and Melanocyrillium sp. Analyses in whole rock samples of residues from water (H2O) and hydrogen peroxide 30% (H2O2) procedures showed similar results when the clay fraction studied was obtained as part of micropaleontological preparation compared with the results from the standard clay mineral preparation method. The clay fraction diffractograms showed that the micropaleontological preparation with H2O and H2O2 caused an increase in the intensity of the quartz reflections compared with untreated samples. Moreover, detailed protocols for organic-walled microfossil preparation and low concentrated acetic and formic acids attacks for mineralized microfossil extraction were presented.

Optimization of the acetic acid method for microfossil extraction from lithified carbonate rocks: Examples from the Jurassic and Miocene limestones of Saudi Arabia

An optimization experiment with different acid concentrations was carried out to assess the use of acid to minimum sustainable limits for the extraction of microfossils from indurated limestones. Two different limestone formations of Jurassic and Miocene ages were tested. Different concentrations of acid ranging from 50 to 100% and processing times varying from 2 to 10 h were tested for optimal recoveries. The acid residue recoveries show a similar trend for both formations. The weight percentage of residue with particle size >1 mm decreased as the acid concentration increased, especially in the 50–80% acid concentration range. On the other hand, the weight percentage of the smallest size particles > 0.063 mm increased as acid concentration increased. This means that the higher concentrations of acid dissolve more of the unnecessary large particles while the foraminifera, which comprise the sand fraction size, are left in the residue. Although higher acid concentrations with longer reaction times yielded better recoveries than with less reaction time, we recommended a 60% concentration of acetic acid and a reaction time of 10 h for optimal recovery of micropaleontological samples in Saudi Arabian carbonate rocks. By lowering the recommended concentration, the consumption of acid is reduced without compromising the recovery of microfossils.•Acetic acid leaching method is applied on two different age limestone samples to extract foraminifera.•Different concentrations of acetic acid are tried and tested, and consensus is made on an optimum concentration of 60% for a submersion time of 10 h.•The sample recoveries are optimal while using this concentration for a time of 10 h.

Successful complete digestion of well lithified shale and extraction of microfossils from Devonian beds in western New York

ABSTRACT: Researchers in paleontological and paleoecological sciences often need complete disaggregation of rock materials for certain lines of investigation. However, complete disaggregation of more lithified sedimentary rock is known to be problematic. A complete shale disaggregation method implementing quaternary ammonium surfactants,widely used in paleontological sciences for poorly lithified shale and mudstone, was successfully used on well lithified Devonian shale in the Appalachian Basin ofWestern New York. Over 50 Devonian gray and black shale samples were collected from multiple localities in western New York (Cashaqua, Rhinestreet, Skaneateles, Windom, and Ludlowville), coarsely crushed, and fully immersed in a quaternary ammonium surfactant until complete disaggregation was achieved (5–14 days); aliquots were run through a series of nested sieves. The sieved sediments contained hundreds of well-preserved microfossils released from the shale: ostracods, dacryoconarids, and previously unreported palymorphs, charophytes, agglutinated foraminifera, miospores, and other microspherules. These microfossils were easily found within disaggregated and sieved samples but were unrecognizable on the shale surface and destroyed in prior investigations of whole rock thin sections. In addition to more traditional approaches, inclusion of this complete rock disaggregation method may assist in a more complete analysis of material, increase our understandings of ancient basin systems and have important implications on our understanding of the paleoecology during the Late Devonian marine biotic crises.

Why loss matters: Reply to the comments of Festi and others on ‘A quantitative comparison of microfossil extraction methods from ice cores’ by Brugger and others (2018)

Why loss matters: Reply to the comments of Festi and others on ‘A quantitative comparison of microfossil extraction methods from ice cores’ by Brugger and others (2018)

Comments on Brugger and others (2018) ‘A quantitative comparison of microfossil extraction methods from ice cores’

ABSTRACTIn our comments, we re-evaluate Brugger and others (2018) Lycopodium/Eucalyptus double marker approach, based on the fact that previous evidence already demonstrated that the batch of Eucalyptus tablets used by Brugger and others (2018) is not suitable for quantitative comparisons as they are characterized by inconsistent pollen concentration. We present clear evidence that the Eucalyptus tablets do feature inaccurate pollen concentrations, and are therefore improper for all quantitative comparisons of microfossil extraction methods. Consequently, the results of the quantitative and qualitative assessment of different pollen extraction methods from ice samples compiled by Brugger and others (2018) are highly questionable due to the use of faulty marker tablets.

A quantitative comparison of microfossil extraction methods from ice cores

ABSTRACTMicrofossil records from ice archives allow vegetation, fire and land-use activity reconstructions on broad spatial scales. Samples typically contain low microfossil concentrations. Therefore, large ice volumes are often needed for palynology. Hence, it is crucial to extract maximum microfossil numbers through appropriate physical-chemical treatments. We compare six methods covering the main water reduction procedures: evaporation, filtration and centrifugation with snow samples. Adding a known number of Lycopodium marker spores prior to sample treatment and a second marker (Eucalyptus) after laboratory processing allows a quantitative microfossil loss assessment during pollen extraction. We applied the best-performing method (average loss of 22%) to high-alpine firn cores from Colle Gnifetti glacier for validation with a natural archive containing extremely low microfossil concentrations. We conclude that samples processed with different microfossil extraction protocols may give different results for pollen concentrations, percentages and ratios between different pollen types, especially if vesiculate conifer pollen is an important pollen assemblage component. We recommend a new evaporation-based method which delivers the smallest and least variable losses among the tested approaches. Since microfossil losses are inevitable during laboratory procedure, adding markers prior to sample processing is mandatory to achieve reliable microfossil concentration and influx estimates.

Palaeozoic ‘conodont pearls’ and other phosphatic micro-spherules

Sub-millimetre-sized phosphatic spherules are often found in acetic acid-insoluble residues produced for microfossil extraction. As they are typically associated with conodont elements and have a similar chemical composition, they are informally known as ‘conodont pearls’. Still, the origin of these micro-spherules has been controversial, and authors have disagreed regarding their mode of formation, or if they are biogenic or not. In this study, an assortment of micro-spherules from several localities and stratigraphical levels were analysed using multiple methods, in an effort to shed light on the origin of these enigmatic objects: ocular investigation with a stereomicroscope, chemical analyses employing energy-dispersive mass spectrometry, imaging through scanning electron microscope, and synchrotron radiation X-ray tomographic microscopy. Collectively, the techniques employed allow for near-complete characterization and description of the study specimens. At least five different groups, or morphotypes, of spherules can be discerned, which differ both in morphological and chemical details. Most specimens are notably spherical and display concentric layers of growth. Several of the specimens have a central nucleus, sometimes with one or more objects located closely together. Internal details and chemistry suggest that the phosphatic spherules probably are of different origin. Thus, the term ‘conodont pearl’ encompasses confusingly similar objects deriving from different organisms and/or processes, and only careful analysis can reveal their individual origin. The only organisms unequivocally associated with (in situ) phosphatic micro-spherules are ceramoporid bryozoans, and the conodonts Cordylodus and Westergaardodina, but the possible function and significance of these objects remain enigmatic.

An integrated microwave technique for releasing microfossils from an indurated bone bed

Bone beds have often been a focus for micropalaeontological study as the high concentration of fossil material allows vertebrate palaeontologists the returns normally experienced only by nannofossil workers and palynologists. It is not always straightforward to release the fossil material within bone beds and the extraction of microfossils from the upper Silurian Downton Bone Bed is particularly challenging. Outlined here is an integrated method for processing this bone bed using liquid paraffin and a microwave oven, and a comparison in terms of quality and quantity with material recovered using other techniques. This integrated method allows for the recovery of microvertebrates, such as thelodonts, and also internal moulds of ostracods, brachiopods and early plant material.

Development of the freeze–thaw processing technique for disaggregation of indurated mudrocks and enhanced recovery of calcareous microfossils

Abstract. Microfossil extraction from indurated mudrocks is widely acknowledged as challenging, especially for foraminifera. Here we report development of the freeze–thaw extraction method through the addition of rapid heating, detergent and ultrasound stages. We use indurated mudrock samples from the Toarcian (Early Jurassic) of Yorkshire, UK to assess the effectiveness and develop the freeze–thaw method. We compare our results from freeze–thaw with those from standard foraminifera processing techniques, including the use of hydrogen peroxide. Processing by freeze–thaw increased the degree of mudrock disaggregation and resulted in no damage or dissolution of foraminifera. Following the freeze–thaw method with treatment in white spirit and sodium hexametaphosphate aided the separation of foraminifera from the disaggregated clays and was twice as efficient as pressure washing. Samples processed with hydrogen peroxide contained damaged microfossils and an under representation of delicate calcareous foraminifera. Many other studies of indurated mudrocks have used hydrogen peroxide to extract foraminifera, and this might have resulted in apparently barren intervals. The freeze–thaw method outlined here provides a low-cost, low-risk and successful method of disaggregating and extracting calcareous microfossils from indurated mudrocks. We anticipate our method may be relevant for other fossil groups and merits further development.

An acid-free method of microfossil extraction from clay-rich lithologies using the surfactant Rewoquat

Marine rocks characterized by high clay content provide excellent conditions for fossil preservation, particularly for organic-walled microfossils such as retiolitid graptolites and chitinozoans. Nevertheless, the phyllosilicate minerals, which constitute the clay component, make microfossil extractions difficult. The problem results from the tendency of phyllosilicates to form aggregates in low pH values, as standard methods of microfossil extraction employ acids for rock digestion. Consequently, the use of acids for clay-rich rocks is often inefficient and time-consuming. We propose a method of rock disintegration using the surfactant Rewoquat and compare it with two commonly applied approaches: digestion in buffered acetic acid and in HCl-HF. Using examples from the Mulde Brick-clay Member from the Silurian of Gotland and the Daleje Shale from the Devonian of the Prague Basin we observed that disintegration in Rewoquat was faster (days) than digestion in acid (months), and allowed to recover calcareous in addition to organic-walled fossils. The yield and preservation was comparably good, except for conodonts, which were strongly etched after using HCl-HF. Retiolitid graptolites recovered using Rewoquat were preserved in 3D and showed a lower degree of fragmentation. The fossil content of the residue obtained using Rewoquat was higher due to dispersion of clay aggregates. For observation of delicate fossils we recommend to coat the sample with the surfactant. Application of Rewoquat can reveal the most delicate forms and growth stages, and thus provide a better insight into the ontogeny, autecology, and body size distributions of a number of fossil groups.

Dating the Tethyan Ocean in the Western Alps with radiolarite pebbles from synorogenic Oligocene molasse basins (southeast France)

For the first time in the Western Alps, radiolarite pebbles collected from Tertiary foreland molasse conglomerates are treated for microfossil extraction and dated. Among forty pebbles collected in the field, seven of them released diagnostic radiolarian assemblages ranging in age from Late Bajocian–Early Callovian to Middle Oxfordian–Early Tithonian. These ages overlap previous biochronological data obtained from in situ localities of the Schistes Lustrés Piemont zone of the French-Italian Alps and triple the number of diagnostic radiolarite samples known so far in this segment of the chain. The diagnostic pebbles are characterized by low grade metamorphism, showing that some eroded thrust-sheets from the oldest parts of the ocean escaped any tectonic burial during the Alpine convergence. Mixing of low and high-grade radiolarites, mafics and ultramafics pebbles implies that various ocean-derived units were exposed. This tectonic scenario involves tight refolding and severe uplift of the Eocene subduction wedge in the Early Oligocene.

A new method of rock disintegration and foraminiferal extraction with the use of liquid nitrogen [LN2]. Do conventional methods lead to biased paleoecological and paleoenviromental interpretations?

An extremely fast, easy, and clean method of rock disintegration and foraminifera extraction from variously lithified porous rocks with the use of liquid nitrogen [LN 2 ] is proposed. This method markedly limits the time of rock disintegration from days to only minutes, is safe for foraminifera, and does not require special chemical labs. In the experiment, the LN 2 method was used to decompose rock samples and simultaneously to extract the foraminifera hidden within. The proposed method disintegrates the rocks to a finer fraction than conventional methods such as the Glauber's Salt method, allowing to collect more smaller planktonic and benthic foraminifera, resulting in marked changes in foraminiferal assemblages e.g., the planktonic/benthic ratio [P/B defined as P/P+B], leading to new conclusions. The comparison between the LN 2 and Glauber's Salt [GS] methods and obtained results are provided. ► Fast and easy microfossil extraction with use of liquid nitrogen is proposed. ► The time needed for rock disintegration is limited from days to only minutes. ► The new method gives more reliable results e.g. P/B ratio. ► New perspectives and possibilities in paleoenvironmental interpretations. ► The method seems to be applicable for variously lithified porous rocks.

Laboratory goals and considerations for multiple microfossil extraction in archaeology

We discuss some recent trends in archaeological microfossil research, including approaches using multiple microfossil data sets (e.g. pollen, phytoliths, starches, and microscopic charcoal). We review eight types of microfossils, with reference to their physical characteristics, their uses in archaeology, and specific requirements for laboratory extraction and microscope viewing. Rather than presenting any single processing protocol, in order to assist individual researchers or laboratories in developing safe, effective, and economical procedures for extracting their microfossils of interest, we provide guidelines based on previous studies from various fields of microfossil research. We articulate the various general goals of extraction and slide-mounting protocols, and tabulate the potentially destructive effects of discrete methodological procedures on individual microfossil types, including raw sample preparation, disaggregation, deflocculation, clay removal, organic and mineral destruction, heavy-liquid flotation, slide-mounting, and light microscopy. Finally, we present two illustrative archaeological case studies: (1) an example of developing a microfossil extraction protocol to accommodate refractory volcanic soils from the Hawaiian Islands, and (2) a discussion of the development of a low-chemical laboratory approach for extraction and interpretation of multiple microfossil types from agricultural and pastoral archaeological sites in an Argentinean high valley.

Late Albian–Coniacian planktic foraminifera and biostratigraphy of the northeastern Caucasus

This paper presents a considerably revised biostratigraphy for Upper Albian through Coniacian pelagic limestone and shale sequences in the northeastern Caucasus region based primarily on planktic foraminiferal distributions. The use of concentrated acetic acid for the extraction of microfossils from the hard limestones has yielded a much more detailed planktic foraminiferal biostratigraphy than has been documented previously. Because of the low latitude location of the study area the high diversity assemblages contain many of the biomarkers used to identify standard Tethyan biozones ranging from the Rotalipora appenninica Zone through the Dicarinella concavata Zone. A key result of this study is the recognition of an apparently continuous Cenomanian/Turonian boundary interval within a laminated, dark marl that is enriched in organic carbon. Extinction of the single-keeled rotaliporids corresponds with the onset of deposition of the laminated marl beds.

Artificial chemical degradation of some extant cyanobacteria with special reference to Precambrian contaminants- A cautionary note-II

The paper is a cautionary note to all Precambrian palaeobiologists, using palaeopalynological techniques for the extraction of microfossils from Precambrian sediments. It deals with problems of contamination in palaeopalynological preparations. The effect of mineralic acids (HCI and HF) on some common cyanobacteria, viz., Microcystis, Nostoc, Oscillatoria, Lyngbya and Scytonema, has been discussed. It has been shown that the extant cyanobacteria after treatment with these mineralic acids show morphological similarities with many Precambrian microfossils. Therefore, there is a need to exercise more vigil while ascertaining the authenticity and affinity of the microfossils recovered through palaeopalynological techniques specially from Precambrian sediments.