Functional interpretation of archaeobotanical data: Making hay in the archaeological record

Hay malting and hay meadows have long been of fundamental ecological, economic and social importance in temperate Europe. A variety of archaeological sources suggests that hay making may date back to the Iron Age, but direct archaeobotanical evidence for this practice is problematic. Past grassland communities are imperfectly represented and preserved in archaeobotanical assemblages, and ancient meadow and pasture communities may not resemble present-day communities in terms of management practices or botanical composition. This paper explores the potential of FIBS (Functional Interpretation of Botanical Surveys) in the archaeobotanical investigation of ancient grassland management. The botanical composition of present-day grassland communities was analysed in terms of functional attributes (e.g. canopy height) relevant to cutting, grazing and habitat productivity. The utility of these attributes for distinguishing between present-day meadow, pasture and unmanaged grassland communities has been evaluated. Similar analyses were performed on archaeobotanical data from Neolithic to post-Medieval northwestern and central Europe. Functional shifts over time, interpreted in the light of the functional analysis of modern grassland, suggest that hay-making was well established by the Iron Age. Avenues are suggested for the refinement and further development of the FIBS methodology in the archaeobotanical investigation of grassland management.     

Determination of the toxicity of faeces of cattle treated with an ivermectin sustained-release bolus and preference trials using a dung fly, Neomyia cornicina

The toxicity of dung from cattle treated with an ivermectin sustained-release bolus was estimated in terms of ivermectin or ivermectin equivalents, using a laboratory bioassay with the dung fly Neomyia cornicina Fabricius (Diptera, Muscidae). The mortalities of flies measured 7 days after feeding for 24 h on dung containing known concentrations of ivermectin (between 0.125 and 1 g ivermectin per gram fresh dung) were compared with the mortalities of insects fed for 24 h on dung from cattle treated 21 days previously with an ivermectin sustained-release bolus. The toxicity of the bolus dung was equivalent to dung containing 0.66 g ivermectin per gram fresh dung. To determine whether insects could differentiate between control dung and dung from bolus-treated cattle, choice-chamber tests were carried out. There was no significant difference in the percentage of females that chose either dung type, suggesting that they were unable to distinguish the dung of bolus-treated cattle from control dung. Results are discussed in relation to the impact that bolus use can have on the insect fauna of cattle dung.     

The first herders in the upper Ebro basin at Los Husos II (Álava,Spain): microarchaeology applied to <Emphasis Type="Italic">fumier</Emphasis> deposits

Fumier deposits are the product of the recurrent use of caves and rock shelters for stabling livestock and the periodic burning of the resulting dung. Their chronologies in the Mediterranean area extend from Neolithic times up to the Bronze Age, but they are scarce in or absent from Iron Age sites. The study of these deposits has provided important information to better understand past livestock and husbandry practices. The archaeological site of Los Husos II, Álava, in the upper Ebro basin, Spain, dating to 6,990–6,770 cal bp, represents one of the earliest pieces of evidence for animal domestication in the Basque Country. The Ebro basin is particularly important, since this was the main route by which the Neolithic economic system spread from the Mediterranean coast to the northern Iberian Peninsula and the western Pyrenees. We present here the results of the study of the fumier deposits from the Neolithic Levels IV to IX of Los Husos II, through analyses of phytoliths, faecal spherulites and ash pseudomorphs. The main goal was to discover the ways in which Neolithic populations in this region adapted these new practices and carried out their activities. The results indicate a constant pattern of keeping animals throughout the study period. The principal component of the livestock diet consisted of wild grasses from the vicinity. The presence of grass inflorescences suggests a diet rich in summer grass. In addition to grasses, dicotyledonous plants were also indicated, both through phytoliths and ash pseudomorphs. Faecal spherulites from herbivores were also noted in the samples and together with phytoliths and ash pseudomorphs they give important information regarding the formation processes of the studied deposits.     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

Pollen selection for Alternaria resistance in oilseed brassicas: responses of pollen grains and leaves to a toxin of A. brassicae

Summary The effects of destruxin B, a host-specific toxin of Alternaria brassicae that causes black spot disease in oilseed brassicas, were studied on in vitro pollen germination and pollen-tube growth of Brassica campestris var brown sarson, B. juncea, B. napus cvs Westar and Cresor, B. nigra and Sinapis alba. Pollen grains of B. nigra, B. juncea and B. campestris were the most sensitive and those of S. alba the least sensitive to the toxin. Effects of the toxin were also studied on the leaves of these species, and the degree of sensitivity of leaves of different species was comparable to that of their pollen grains. The results on the responses of pollen grains as well as leaves to the toxin are in agreement with the degree of susceptibility/resistance of these species to A. brassicae reported in the literature, indicating that the genes imparting susceptibility/restistance are expressed in the pollen, a prerequisite for pollen selection. Results are also presented which show that the toxin fed to the cut end of isolated inflorescence axis is readily taken up by the developing pollen and results in the inhibition of germination of susceptible pollen. This technique offers a simple and effective method for application of selection pressure to eliminate pollen grains susceptible to the toxin from effecting fertilization.     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

Mentor pollen: Possible role of wall-held pollen growth promoting substances in overcoming intra- and interspecific incompatibility

Recent studies have shown that certain normally incompatible, interspecific combinations in the genus Populus, and intraspecific combination in Cosmos bipinnatus can be made compatible by mixing irradiation- or chemically-killed compatible pollen (mentor) with normal incompatible pollen at the time of pollination. Knox and associates have proposed that in the sporophytic system of incompatibility the compatible mentor pollen provides the so-called specific recognition substances to the incompatible pollen thereby making the latter compatible. In the present experiments mentor pollen was used in attempts to overcome both intra- and interspecific incompatibility in the genus Nicotiana which has a gametophytic intraspecific incompatibility system. Although not denying that recognition substances specific to pollenpistil incompatibility reactions are present in pollen walls, the results suggest that a role of mentor pollen in overcoming incompatibility may be to provide extra free pollen growth promoting substances (PGS). These substances are apparently able to promote just sufficient further growth to allow some fertilizations by certain pollen tubes with inherently weak expression of incompatibility.The present proposed PGS-Hypothesis also elucidates the physiological basis of the widely-held relationship found between genetic systems of self-incompatibility and sites of pollen inhibition.     

Interaction of PRK1 Receptor-like Kinase with a Putative elF2B β-Subunit in Tobacco

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast -subunit of translation initiation factor 2B (eIF2B-), which was designated NeIF2B. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2B were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2B, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2B and PRK1 proteins. Despite high sequence homology between NeIF2B and its yeast counterpart, the NeIF2B cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-, when transferred into the mutant strain.     

Are modern savannas degraded forests?-A Holocene pollen record from the Sudanian vagetation zone of NE Nigeria

A pollen record from a crater lake (Lake Tilla, 10°23N, 12°08E, c. 700 m asl) in the Sudanian zone of northeast Nigeria provides evidence for the persistence of woodland savanna throughout the Holocene. Wetter conditions from c. 10,000 B.P. to c. 6800 B.P. enabled the establishment of a dense Guinean savanna, though the occurrence and rapid spread of the montane elementOlea hochstetteri indicates cool climatic conditions prior to c. 8800 B.P. Patches of closed dry forest may have existed, but never completely displaced the savanna vegetation. Grass fires were frequent throughout the Holocene and were probably important in promoting the open character of the vegetation. From c. 6800 B.P. onwards a gradual floristic change from a Guinean to a Sudano-Guinean savanna and a lowering of lake levels point to drier environmental conditions, which intensified around 3700 B.P. Human impact might have caused increasing sedimentation rates from c. 2500 B.P. onwards. The pollen diagram of Lake Tilla reflects a history of the savanna which appears to have been primarily controlled by climatic changes. The lack of unambiguous pollen indicators might be the reason why human activities remain palynologically hidden even for the late Holocene.     

Influence of heat shock on seed germination of plants from regularly burnt savanna woodlands and grasslands in Ethiopia

The effect of heat shock on the germination of seeds of 21 plant speciesfrom fire-prone wooded savanna ecosystems in western Ethiopia was analysed inorder to examine the possible implications of fire upon plant regenerationafterthis disturbance. Seeds were subjected to 6 different heat intensities (20, 60,90, 120, 150 and 200°C) for 1 or 5 minutes, in ordertosimulate the situation in the upper soil layers or on the soil surface duringfires. Germination tests were carried out in pots in a greenhouse over 20weeks.After 9 weeks no more seedlings emerged. There was wide interspecific variationin the responses of seeds to the different treatments. In all species,germination was significantly affected by the temperature treatment level.Shortexposure of seeds to high temperatures generally stimulated germination whereasprolonged exposure reduced seed germination. However, some species eventolerated 5 min treatment at 200°C. Seedheat resistance was positively correlated with seed length and mass among thespecies. Hence, production of large seeds with protective tissues promotessurvival in fire-prone savanna areas. Also, the seeds of some species showedboth a low and a high temperature optimum which ensures that at least someseedsgerminate in the absence of fire, but also that viable seeds still remain ifsubsequent late fires kill emerging seedlings. Frequent and light burning inwooded savanna grasslands seems to stimulate and enhance germination of most ofthe studied plant species.     

Contemporaneous Alnus decline and the beginning of Iron Age cultivation in pollen stratigraphies from southern Finland

Declines in Alnus coinciding with the first signs of Iron Age (a.d. 0–1150) human activities were found in the pollen stratigraphies of five small lakes in southern Finland. One lake did not show a clear minimum. Three of the lakes were investigated with close-interval analyses which showed that the Alnus minimum lasted for several centuries. The results were compared with 41 previously published pollen diagrams with evidence of Iron Age human activity from southern Finland. These diagrams were classified in three ways: (1) showing no Alnus minimum; (2) cases where a minimum was unclear; (3) showing a clear minimum in Alnus. The different types were found randomly scattered around southern Finland suggesting that Alnus minima were a local phenomenon. In most cases the Alnus minimum took place between ca. a.d. 600 and ca. a.d. 1000, a.d. 1300 being the latest date for the end of the minimum. The results do not suggest a pathogen outbreak over the entire area. The beginning of the minimum clearly coincides with the onset of Iron Age anthropogenic activities suggesting that these were the probable cause. Pollen analysis provides little information as to why trees were felled thus archaeological evidence is needed. However, the Alnus decline may prove a new and useful indicator of the onset of Iron Age anthropogenic activity in pollen diagrams.     

Attractiveness of baits to dung beetles in Brazilian savanna and exotic pasturelands

The Brazilian savanna is the second largest ecosystem in Brazil. It is also one of the most endangered, with only 20% of its habitat remaining unchanged. Agriculture and livestock have been indicated as the main agents of destruction of the Brazilian savanna. Brazilian livestock, for example, is the main reason for cultivation of exotic grasses such as Urochloa spp. (from Africa). Dung beetles are widely used in ecological assessment, mainly because they are recognized as bioindicators of environmental changes. Therefore, efficient sampling is required for any research involving the biodiversity of this group. In order to mitigate the lack of information on efficiency of the attractiveness of baits in the endangered Brazilian savanna and in exotic pasturelands, we sampled dung beetles in four native patches of the Brazilian savanna and in four areas of pastures with Urochloa spp. Dung beetles were captured with pitfall traps baited with carcass, cattle dung, human feces and pig dung, with a total sampling effort of 384 traps. We sampled 7544 individuals belonging to 43 species and 18 genera of dung beetles. Thirty‐eight species were collected in the Brazilian savanna and 24 species in exotic pastureland. In both ecosystems traps baited with human feces sampled greater abundance and species richness of dung beetles when compared with the other three baits used. Our results showed that human feces is a reliable, easy and inexpensive bait to sample greater abundance and species richness of generalist dung beetles in both native and exotic habitats, with clear structural differences.     

Variation in Ambrosia pollen concentration in Southern and Central Poland in 1982–1999

The aim of the study was to investigate theAmbrosia pollen concentrations inselected Polish cities and for Kraków torelate it to some meteorological factors.Sampling was carried out in Kraków in1982–1997 and in Rabka in 1992–1996 with theuse of the gravimetric method. In Zakopane,Kraków, Ostrowiec witokrzyski,Warszawa and Pozna in 1995–1996 both thegravimetric and volumetric methods (Burkardtrap) were employed. In Kraków themonitoring has been performed since 1994 usingthe volumetric method. The results show theragweed pollen presence in August and Septemberwith the tendency to appear more frequently inAugust in some years. In Kraków (1994–1999)Ambrosia pollen was found either in thelast two weeks of August or in the first twoweeks of September which seems to be a regularand repeatable pattern every two years.Seasonal fluctuations of Ambrosia pollenconcentration do not show a clear increasingtendency except at Warszawa and Ostrowiecwitokrzyski in 1996 and at Krakówin 1999. Percentage of Ambrosia pollen inannual sums of total pollen is very low anddoes not exceed 1% except at Ostrowiecwitokrzyski in 1996 (1.2%) and atKraków in 1999 (2%). For Kraków theanalysis of some meteorological factors (Tmax,Tmin, precipitation, wind direction) wasperformed. High temperature and lack of rain orlow precipitation correlate well with ragweedpollen concentrations. During the Ambrosia pollen seasons ESE, E, S, SE, WSW, SWwind directions prevailed which could suggest along-distance transport from Ukraine, the CzechRepublic, Slovakia and also from Hungary, one ofthe most ragweed-polluted countries.     

Tracing microfossil residues of cereal processing in the archaeobotanical record: an experimental approach

Interdisciplinary methodological approaches are fundamental for studying tool use and crop processing patterns in the archaeological record. Many archaeological studies of plant microfossil evidence, primarily those of phytoliths, starch grains and pollen, are concerned with processing methods which can be replicated through experimentally produced plant residues. However, most of these studies rely on crop identification through the presence or absence of such microfossils while giving little or hardly any weight to taphonomy and formation processes, which are critical for interpreting archaeological contexts. An investigation of experimentally produced phytolith and pollen assemblages provides the opportunity to evaluate the impact of cereal processing on both microfossils. Controlled experiments were conducted at the Museum of Menorca, Balearic Islands, Spain, for assessing microfossil taphonomy using Iron Age Talayotic tools and Hordeum vulgare (hulled barley) grown nowadays on the island. For dehusking, a sandstone mortar and a wooden pestle were used outdoors, whereas grinding took place indoors using a limestone quern and handstone. The results indicate that the size of multicellular or anatomically connected phytoliths decreases as a result of mechanical degradation suffered through processing activities, whereas the proportion of cereal pollen grains increases through these processes. Additionally, experimental samples from dehusking and sieving provided abundant evidence of floral bracts, and also of other plant parts and even different plant species, such as phytoliths from leaves and stems and non cereal pollen taxa, which were also to be found on the surfaces of the ground stone tools. These findings highlight the importance of integrating different lines of microfossil evidence and taking into account formation and taphonomic aspects, as well as the value of experimentally produced data for a better understanding of tool use and crop processing.     

Preliminary studies of the impact of excreted N on cycling and uptake of N in pasture systems using natural abundance stable isotopic discrimination

The possibility of using natural abundance techniques to determine N transformations and flows after deposition of cattle dung has been examined. These preliminary results showed that ¹⁵N in dung was greater than in plants growing in association with particular pats. This, and other observational information, indicated that dung pats of different ages were being examined. There were significant variations in plant ¹⁵N signatures within and between species grown in association and away from the dung. It was probable that variation in plant ¹⁵N was brought about by changes in soil mineral N pools after transfer of N derived from the dung. This resulted in different ¹⁵N signatures in Trifolium repens (because of changes in N utilization from soil or atmospheric pools), in Lolium perenne (because of changes in ¹⁵N in soil mineral N), but not in Ranunculus repens (because the majority of active roots were outside the range of immediate influence of the deposited dung). The differences in ¹⁵N allowed the development of hypotheses for changes in soil N pools and the acquisition of N by plants from soil, dung or atmospheric sources.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

The pollination biology ofHibbertia stricta (Dilleniaceae)

The exines of pollen grains ofHibbertia stricta (DC.)R. Br. exF. Muell. (Sect.Pleurandra) wear an oily, yellow pollen coat that stains positively for lipids. The pollen is collected by asocial bees, exclusively. The most common floral foragers are members of the genusLasioglossum (subgenusChilalictus;Halictidae) and they harvest pollen via thoracic vibration. As these bees cling to the inflated anthers their pollen smeared bodies come in contact with either of the two wet, nonpapillate stigmas. The stigmas respond positively to cytochemical tests for the presence of esterase immediately following expansion of the corolla, indicating the effective pollination period. The foraging patterns of the bees are narrowly to broadly polylectic. AsH. stricta flowers are nectarless, it is not surprising that bees bearing mixed pollen loads always carry the pollen of at least one nectariferous, coblooming plant. The pollination biology ofH. stricta is compared with otherHibbertia spp. and with pollen flowers in general.     

Dissection of a pollen-specific promoter from maize by transient transformation assays

We have previously reported the isolation and characterization of a gene (Zm 13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm 13 5 flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5 regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the -glucuronidase (GUS) gene under the control of various sized fragments of the Zm13 5 flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from –100 to –54, while other sequences which amplify that expression reside between –260 and –100. The replacement of the normal terminator with a portion of the Zm13 3 region containing the putative polyadenylation signal and site also increased GUS expression. While the –260 to –100 region contains sequences similar to other protein-binding domains reported for plants, the –100 to –54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.