JPT No. 9 – How to mount an inexpensive sieving lab

Ricardo Araújo¹,², Carlos Natário¹, Matthew Pound³,⁴

1 – Museu da Lourinhã, Rua João de Luís de Moura, 95, 2530-158 Lourinhã, Portugal.
2 – Huffington Department of Earth Sciences, Southern Methodist University, PO Box 750395, Dallas, Texas, 75275-0395, USA.
3 – School of Earth and Environment, University of Leeds, Leeds, LS2 9JT, United Kingdom.
4 – British Geological Survey, Keyworth, Nottingham, NG12 5GG, United Kingdom.

ABSTRACT 

We present a new sieving technique designed to recover microfossils from mudstones, clays and poorly consolidated sediments. This new technique is designed to be inexpensive, use the minimal amounts of chemicals and recover well preserved microfossils. The inexpensive nature of this methodology makes it suitable for reconnaissance studies, where finances may be limited. Not using large amounts of chemicals helps to protect the fossils and the environment. Investigations on the Jurassic Lourinhã Formation, Portugal yielded a diverse microfossil assemblage including; archosaur teeth, lizard jaws, amphibian jaws, fish remains, ostracods and charcoal. Such a diverse fossil recovery shows the technique is suitable for painstaking palaeoecological studies as well as reconnaissance work.

INTRODUCTION

The use of confocal laser scanning microscopy (CLSM) for the study of inclusions in amber is a recent development. Böker & Brocksch (2002) produced a series of images of insects in Baltic amber using CLSM and identified the potential for 3D imaging of minute detail of taxonomically important morphological structures such as the mandibles and genital organs. Since then, however, very little has been published on CLSM analysis of amber inclusions despite the apparent benefits.

Sieving methods for microvertebrate recovery have rarely (Mateus et al. 1997) been applied to the Lourinhã Formation. The Lourinhã Formation is comprised of fossiliferous fluviodeltaic deposits that outcrop extensively in the Lusitanian Basin, Western Portugal. This formation is mainly composed of intercalated sandstone channels with extensive alluvial mudstone layers (Hill 1989). Within the extensive mudstone layers most fossils including many large vertebrates (Antunes and Mateus, 2003) and microfossils are found (e.g. Ramalho 1967). Microvertebrate fossil faunas have been collected for many years in the Lourinhã Formation, mainly by surface collecting. However, in 2008 the Museum of Lourinhã started a systematic sieving campaign to better understand the overall fauna of the Lourinhã Formation. 

Some of the first microvertebrate finds in Portugal were discovered in the Guimarota mine in 1960 by palaeontologists from the Freilicht Universitat, Berlin (Krebs, 2000). The first account of sieving methods being used in Portugal were published by Kühne (1968), using a constant flow of water and sieves incorportated on a metal barrel. Sieving was applied at the Paimogo theropod embryo nest site (Mateus et al. 1997) from 1994 to 1996, in the search for embryo bones and eggshells (Mateus, 1998) and was also occasionally applied to the Porto das Barcas fossil site, but without much success.

Precise geographical information of sieving sites in the Lourinhã Formation was acquired using GPS coordinates, stratigraphic information and a measured section was acquired by plotting the site photograph with detailed geologic annotation. Samples were taken using a pick axe at 30 – 40cm stratigraphic intervals and within 1 – 2m horizontal spacing. This systematic sieving campaign, applied to the Lourinhã Formation for the very first time, is being used to investigate and assess the composition and diversity of the microfossil fauna.

The first use of sieving in the search of microvertebrate remains was performed ca. 1847 by Plieninger, in Germany (McKenna et al. 1994). Early methods of sieving were also used by Moore in England (1867) and later by Wortman and Brown in the United States (1891).  Sieving became well implemented in the palaeontological community after Hibbard (1949) reported using the technique to collect Cenozoic mammal fossils from an unconsolidated sandstone matrix (McKenna et al., 1994). Hibbard introduced the use of screen boxes (wooden boxes with a brass mesh). However, this method requires having water near the work site, is laborious, and requires a large staff (Ward 1984). McKenna (1962; 1965) provided further insight into the screen box technique and proposed a standard model using manufactured rectangular wooden boxes and a regular size steel mesh. Both McKenna’s and Hibbard’s techniques are field-oriented and require the presence of nearby water. McKenna simply optimized Hibbard’s technique by processing a larger quantity of matrix using more screen boxes (almost 300, compared to Hibbard’s dozen), and adapting the method to the constraints of particular sites. Grady (1979) described a new method using mosquito nets instead of the classical material of screen boxes with brass mesh, providing a more field-oriented  method  with easily transported equipment (Ward, 1984).

In contrast, the method reported in this paper is similar to other laboratory-oriented techniques. Described in further detail by Kühne (1971) and Krebs (2000), the “Henkel technique” (Henkel, 1966) is a laboratory-oriented technique that was used for more than a decade during the time that microvertebrates were being collected from the Guimarota Mine. This technique is a static sieve method making use of a jet of water that passes through a barrel with a 500µm -mesh on the side. Freudenthal (1976) developed a table technique. This “table” stood 1m high and used a 500µm mesh as a replacement for the table top.  Solid side bars were attached in order to avoid sediment loss.  A jet of water was used to wash the sediment through the table top. 

The methods above described provide excellent guidance for new field researchers, but due to peculiarities of each site/investigation, modifications to the methods may be required.  Aspects such as the location of the fossiliferous horizon (i.e. remoteness), budget and laboratory conditions (i.e. pre-existing infrastructures) can hinder recovery of specimens in an identifiable condition. The methodology here described does not aim to process vast amounts of sediment, as others can. Instead, it does allow individual horizons to be processed without the potential of mixing sedimentary beds. When a sedimentary bed is of limited vertical extent the field-orientated techniques require extensive excavation of the target horizon, losing possible valuable horizons, or mixing multiple sedimentary layers. This can hinder the investigation of fine scale ecological, climatological and evolutionary patterns.

JPT No. 8 – Using Confocal Laser Scanning Microscopy To Image Trichome Inclusions In Amber

Neil D. L. Clark¹ and Craig Daly²

1- Hunterian Museum, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK  – Corresponding author email: nclark@museum.gla.ac.uk

2- Neuroscience & Biomedical Systems, Faculty of Biological and Life Science, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK

ABSTRACT 

Confocal laser scanning microscopy (CLSM) is an analytical technique usually applied to biological and medical samples. It is used to produce high resolution in-focus three dimensional images of thick sections by targeted fluorescence. Trichomes held in amber fluoresce in the far red range whereas amber fluoresces in the ultraviolet. This allows the trichomes to be resolved easily from the amber by CLSM. Samples of amber from two regions were selected for analysis. Baltic amber (Eocene) is well known for its trichome inclusions which have, in the past, been used as a diagnostic feature of that amber. Mexican amber (Middle Miocene) from Simijovel, Chiapas, Mexico also contains abundant trichomes. Samples of amber from both these locations were successfully imaged and re-constructed in 3D using CLSM. This technique enables detailed analysis of the trichome structure without damaging the sample.

RESUMO [in Portuguese]

A microscopia confocal de scanning laser é uma técnica analítica que é geralmente aplicada em amostras biológicas e médicas. Esta técnica produz imagens tridimensionais em foco de alta resolução de secções espessas por fluorescência. Os tricomas aprisionados no âmbar fluorescem na zona mais distante do espectro visível do vermelho enquanto que o âmbar fluoresce no ultravioleta. Isto permite identificar facilmente os tricomas do âmbar por microscopia confocal de scanning laser. Amostras de âmbar nas duas regiões foram seleccionadas para análise. O âmbar do Báltico (Eocénico) é conhecido pelas suas inclusões de tricomas que foram já usadas como características diagnóstico para este tipo de âmbar. O âmbar do México (Miocénico médio) de Simijovel, Chiapas, México também contém tricomas em abundância. Amostras destas duas localizações foram reconstruídas com sucesso em 3D usando microscopia confocal de scanning laser. Esta técnica permite efectuar análises detalhadas das estruturas dos tricomas sem prejuízo da amostra.

INTRODUCTION

The use of confocal laser scanning microscopy (CLSM) for the study of inclusions in amber is a recent development. Böker & Brocksch (2002) produced a series of images of insects in Baltic amber using CLSM and identified the potential for 3D imaging of minute detail of taxonomically important morphological structures such as the mandibles and genital organs. Since then, however, very little has been published on CLSM analysis of amber inclusions despite the apparent benefits.

Another study using this technique, amongst other microscopic techniques, looked at some Spanish amber from Álava (Ascaso et al. 2003, 2005). The study looked at a protozoan with fungal hyphae trapped in amber and produced a 3D image based on a series of optical sections recorded by the CLSM. It is perhaps surprising, considering the detail and quality of the images that can be produced, that more studies have not incorporated this technique.  More recently, Speranza et al. (in press) have used light microscopy, CLSM & widefield fluorescence microscopy to image microscopic fungi embedded in amber, thus confirming the benefits of a ‘fluorescence’ approach. 

Trichomes are present in the vast majority of angiosperms and have been considered for some time to be of importance in comparative systematic studies (Theobald et al.  1979). They have an important role as defensive structures, especially in repelling phytophagous insects (Levin 1973). There is often more than one trichome type on any one taxon, but certain types may be more common to one taxon than another (Theobald et al. 1979). The taxonomic value of trichomes is therefore limited, but the Baltic trichomes are not inconsistent with them being from a type of oak.  

In this study, certain elements of the microflora are examined. The “stellate hairs”, or trichomes, are common in Baltic amber and have been considered as a characteristic of this type of amber (Weitschat & Wichard 2002). These trichomes are found associated with, and attached to, the male oak flowers and are therefore thought to belong to oak also (Weitschat & Wichard 2002) although there may be more than one type of trichome present. During this study samples from Chiapas, Mexico were also examined and were found to contain abundant trichomes. No previous record of trichomes in Mexican amber has been found in the literature.

Weitschat and Wichard (2002) describe the ‘stellate hairs’ found in Baltic amber as structures that develop on the flower and leaf buds of  the oak that are shed in great numbers every year. They also state that no studies have been able to clarify the origin or their significance for amber.

The interpretation of what the inclusions in Baltic amber represent in terms of their ecology has been the subject of much debate as many of the insect inclusions suggest a wide range of biotopes (Weitschat and Wichard 2002). Even individual pieces of amber seem to contain species from what would currently be both temperate and tropical regions (Weitschat 1997). This may suggest that species represent within the amber could have had a wider range then than their extant relatives (Weitschat and Wichard 2002).

In the present study we present a technique which uses CLSM and 3D imaging to analyse the structure of trichomes.

JPT No. 7 – The Setup, Use And Efficacy Of Sodium Polytungstate Separation Methodology With Repsect To Microvertebrate Remains

Jonathan S. Mitchell^1,2 and Andrew B. Heckert¹

1- Department of Geology, Appalachian State University, 572 Rivers Street, Boone, North Carolina, USA

2- Committee on Evolutionary Biology, University of Chicago, 5801 South Ellis Avenue, Chicago, IL 60637-1546; mitchelljs@uchicago.edu (current address)

ABSTRACT 

Concentrated deposits of small remains from vertebrates, termed microvertebrate sites or vertebrate microsites, are a unique and detailed source of information about the history of life. Collecting fossils from these sites, however, presents unique challenges. The most time consuming, and thus most deterring, aspect by far is the separation of the fossils from the sediment. This study attempts to quantify to what extent the use of sodium polytungstate (=sodium metatungstate, Na6H2W12O40, abbr. SPT) filtration increases fossil concentration, how quickly fossils sink in SPT solutions, and what is a good working density for SPT.  We do this by generally following the methodology set out by previous authors, although with some substantial modifications, on an Upper Triassic deposit dominated by clay minerals and lithic fragments, as well as on a second, smaller quartz sand dominated microsite. We also provide a revised and detailed guide with our modifications to former practices and our recommendations to other workers interested in creating a SPT laboratory, including the strong advisory to work over thin plastic sheets, as SPT can react with metal and adheres strongly to glass when it crystallizes.

Our experiments have shown a significant improvement in fossil concentration (from ~2% of the clasts being fossils to ~19%) at the main site, with a sample from the other site showing the treated concentrate as 25% fossil. We have also found very few fossils in the float (<0.5%), but noticeable rates of fossil loss in SPT solutions above ~2.80 g/mL (up to 16%).  Further, we have found that 2.75 g/mL is a good working density for several lithologies, as it is high enough to float most rock, low enough to sink most fossils, and low enough to be manageably maintained. SPT has, in processing one particularly rich site, saved many person-hours that otherwise would have been spent picking through less concentrated sediment.

INTRODUCTION

Studies of microvertebrate fossils (or vertebrate microremains) are becoming increasingly common (Sankey and Baszio, 2008).  Despite providing a wealth of information about past environments and ecosystems, microvertebrate studies are stymied by the difficulty of collection. The process of collecting and isolating large numbers of what are, by definition, tiny and potentially fragile fossils can be extremely time consuming and tedious. Methods have been developed to expedite this process (Cifelli et al 1996:18, Wilborn 2009), yet the fundamental methodology remains extremely similar to its original construction (Hibbard 1949). Further, no one to date has quantified the efficacy of these methods for vertebrate paleontology (though see Bolch, 1997 for dinoflagellates, Krukowski, 1988:315 for conodonts, Murray and Johnston, 1987:319 for heavy minerals in sediments, and Munsterman and Kerstholt, 1996 for palynological experiments).  After a site has been located, it is typically surface collected, then excavated, with vast quantities of sediment being taken away. These bags of sediment are then screen-washed in an attempt to remove as much clay and fine silt, while simultaneously retaining as many fossils, as possible.  After screen-washing, there typically remains a significant volume of concentrate, which is usually composed primarily of non-fossil clasts. After this step, a researcher, preparator or volunteer must go through the screen-washed concentrate one pinch of sediment at a time under a light microscope, isolating and removing individual fossils. These standard techniques for recouping micro-vertebrate  remains from concentrate are extremely time intensive and often dependent on an extensive time investment by students or volunteers (Hibbard, 1949, Grady 1979).

Inevitably, there will be fossiliferous concentrate that needs to be hand picked. The advantage of heavy liquid separation techniques is that they reduce the amount of unnecessary (nonfossiliferous) sediment that needs to be picked through. Traditionally heavy liquid separation was often accomplished using bromide liquids, with their extremely toxic nature representing a significant drawback (Cifelli et al., 1996:17, Murray and Johnston, 1987:317, Murry and Lezak, 1977:17). Murray and Johnston (1987:319) compared SPT to tetrabromoethane (TBE) and found no significant difference for sedimentological applications in the final product, noting only cost and viscosity (concurrent with Cifelli et al., 1996:17-18, though see Jeppsson and Anehus, 1999:57 and below for explanations of this discrepancy) as drawbacks to SPT.

Heavy liquid concentration, regardless of the chemicals used, makes picking both easier and more enjoyable (finding lots of fossils instead of few fossils per unit volume). This also maximizes research time by speeding up fossil recovery. The heavy liquid discussed here, sodium polytungstate (=sodium metatungstate, Na6H2W12O40, abbr. SPT) can be purchased dry and dissolved in deionized water to any desired density from 2.00 g/mL to 3.10 g/mL.

Tungsten compounds have been found to be safe in general (Kazantzis,1979), and sodium polytungstate, unlike bromides and kerosene, is generally regarded as safe unless ingested or applied to the eye (Cifelli et al., 1996:17 and many references therein, also see the Material Safety Data Sheet [MSDS, linked in references] or equivalent safety documentation). Further, sodium polytungstate can be reused continually, assuming it is taken care of properly. It is however, quite expensive (>$200 per 0.1kg), and traditionally difficult to obtain (though the Internet has reduced that problem, as a simple Google® search will reveal). Further, we followed the recommendations of Callahan (1987:765) in using bleached coffee filters instead of filter paper (contra Murray and Johnston, 1987:318) as they appear to speed recovery, but they also seem to have allowed clays to enter and discolor the SPT (though no other side effects have been confirmed, they may have absorbed some of the SPT as a precipitate, see McCarty and Congleton, 1994:198). Six et al. (1999) describe a process of cleansing SPT of organic contents by percolation through a column of activated carbon, and similar methods may work for the removal of clay, though we did not test this, and Murray and Johnston (1987:317-319) and Callahan (1987:765) both argue that laboratory-grade filter paper is enough. Yet as a possible (though unlikely) consequence of clay contamination (clay from a previously separated site contaminating future sites’ fossils) we advise caution in performing geochemical analyses on SPT separated fossils without heavily rinsing them until further studies on the solution’s effects and the efficacy of clay removal are performed.

Despite these modest drawbacks, SPT still provides a powerful tool for the paleontologist/preparator’s arsenal, as we found it easy to use, efficient, and very effective (see below). The ability to continually reuse it, as well as its speed and efficacy, make it cost effective in the long run, albeit a rather large initial investment is required. Here we outline the materials we recommend for a sodium polytungstate separation laboratory, the methods of separation, and the efficacy of the system.