The role of pollen in the changing environmental conditions of Scots pine

Rapid change in climate is challenge for the adaptation of forest trees in the future. In wind pollinated tree species pollen mediated long distance gene flow may provide alleles that are (pre)adapted to a future climate. In order to examine the long distance pollen flow in Scots pine (Pinus sylvestris L.), we measured the amount and viability of airborne pollen and flowering phenology in central, northern, and northernmost Finland during four years. Viable airborne pollen grains were detected during female flowering and before local pollen shedding in all study sites. The situation when there was nonlocal pollen in the air lasted from one to four days depending on the year and study site. The amount of nonlocal airborne pollen varied also between years and study sites, the total amount of nonlocal viable pollen in the air was 2.3% from all detected viable pollen grains. The effect of pollen origin on seeds siring ability was studied with artificial pollination experiments. Pollen genotypes originating from southern Finland sired 76% and 48 % of the analysed seeds in competition studies where both pollen origin were introduced simultaneously into the female strobili. We examined the importance of arrival order of pollen grains in to the strobili in a study where pollen genotypes of different origin were introduced in two hours interval. Northern genotypes sired 76% of the analysed seeds when it was injected first, but in the "southern first" experiment both pollen types sired equal amount of seeds. The first pollen grain in the pollen chamber do not always fertilizes the ovum, instead there likely is more complex way of competition between pollen grains. To examine chemically mediated pollen-pollen interactions we conducted in vitro germination experiment where different pollen genotypes had chemical but not physical contact. Both positive and negative effects of interactions were found. We found highly negative effects in germinability of northern pollen grains when they were germinating with southern pollen, and increase in the germinability of southern pollen. There were no variation in the size of the dry pollen grains between pollen origins, and minor variation between different genotypes. After hydration and germination northern pollen grains were larger than southern pollen. Pollen genotypes having high hydration rates had low germinability and tube growth rate, however, germinated pollen grains were larger in size than nongerminated. This supports the suggestion that the early germination and growth of pollen tube is dependent on pollen storage materialsand less dependent on water intake and hydration. Long distance pollen movements and good competition ability of southern pollen makes gene flow possible, although rising temperature and timing of pollen movements may affect pollen competition and the amount of gene flow.

Heat stress effects on grain sorghum productivity-biology and modelling

Heat stress can cause sterility in sorghum and the anticipated increased frequency of high temperature events implies increasing risk to sorghum productivity in Australia. Here we summarise our research on specific varietal attributes associated with heat stress tolerance in sorghum and evaluate how they might affect yield outcomes in production environments by a crop simulation analysis. We have recently conducted a range of controlled environment and field experiments to study the physiology and genetics of high temperature effects on growth and development of sorghum. Sorghum seed set was reduced by high temperature effects (>36-38oC) on pollen germination around flowering, but genotypes differed in their tolerance to high temperature stress. Effects were quantified in a manner that enabled their incorporation into the APSIM sorghum crop model. Simulation analysis indicated that risk of high temperature damage and yield loss depended on sowing date, and variety. While climate trends will exacerbate high temperature effects, avoidance by crop management and genetic tolerance seems possible.

Release of Bet v 1 from Birch Pollen 1 from 5 European 2 Countries. Results from the HIALINE Study

Exposure to allergens is pivotal in determining sensitization and allergic symptoms in individuals. Pollen grain counts in ambient air have traditionally been assessed to estimate airborne allergen exposure. However, the exact allergen content of ambient air is unknown. We therefore monitored atmospheric concentrations of birch pollen grain and the matched major birch pollen allergen Bet v 1 simultaneously across Europe within the EU-funded project HIALINE (Health Impacts of Airborne Allergen Information Network). Pollen count was assessed with Hirst type pollen traps at 10 l/min at sites in France, United Kingdom, Germany, Italy and Finland. Allergen concentrations in ambient air were sampled at 800l/min with a Chemvol high-volume cascade impactor equipped with stages PM>10μm, 10 μm>PM>2.5μm, and in Germany also 2.5 μm>PM>0.12μm. The major birch pollen allergen Bet v 1 was determined with an allergen specific ELISA. Bet v 1 isoform patterns were analyzed by 2D-SDS-PAGE blots and mass spectrometric identification. Basophil activation was tested in an FcεR1-humanized rat basophil cell line passively sensitized with serum of a birch pollen lmptomatic patient. Compared to 10 previous years, 2009 was a representative birch pollen season for all stations. About 90% of the allergen was found in the PM>10μm fraction at all stations. Bet v 1 isoforms pattern did not varied substantially neither during ripening of pollen nor between different geographical locations. The average European allergen release from birch pollen was 3.2 pg Bet v 1/pollen and did not vary much between the European countries. However, in all countries a >10-fold difference in daily allergen release per pollen was measured which could be explained by long range transport of pollen with a deviating allergen release. Basophil activation by ambient air extracts correlated better with airborne allergen than with pollen concentration. Although Bet v 1 is a mixture of different isoforms, its fingerprint is constant across Europe. Bet v 1 was also exclusively linked to pollen. Pollen from different days varied >10-fold in allergen release. Thus exposure to allergen is inaccurately monitored by only monitoring birch pollen grains. Indeed, a humanized basophil activation test correlated much better with allergen concentrations in ambient air than with pollen count. Monitoring the allergens themselves together with pollen in ambient air might be an improvement in allergen exposure assessment.

Do Urban Canyons Influence Street Level Grass Pollen Concentrations

In epidemiological studies, outdoor exposure to pollen is typically estimated using rooftop monitoring station data, whilst exposure overwhelmingly occurs at street level. In this study the relationship between street level and roof level grass pollen concentrations was investigated for city centre street canyon environments in Aarhus, Denmark, and London, UK, during the grass pollen seasons of 2010 and 2011 respectively. For the period mid-day to late evening, street level concentrations in both cities tended to be lower than roof-level concentrations, though this difference was found to be statistically significant only in London. The ratio of street/roof level concentrations was compared with temperature, relative humidity, wind speed and direction, and solar radiation. Results indicated that the concentration ratio responds to wind direction with respect to relative canyon orientation and local source distribution. In the London study, an increase in relative humidity was linked to a significant decrease in street/roof level concentration ratio, and a possible causative mechanism involving moisture mediated pollen grain buoyancy is proposed. Relationships with the other weather variables were not found to be significant in either location. These results suggest a tendency for monitoring station data to overestimate exposure in the canyon environment.

Seasonality of modern pollen and sediment deposition in an estuarine context: the Severn Estuary Levels, southwest England

Recent sedimentological and palynological research on subfossil Holocene banded sediments from the Severn Estuary Levels suggested seasonality of deposition, registered by variations in mineral grain-size and pollen assemblages between different parts of the bands. Here we provide data that strengthen this interpretation from sampling of modern sediments and pollen deposition on an active mudflat and saltmarsh on the margin of the Severn Estuary, and comparison with a vegetation survey and contemporary records of climate, river and tidal regimes. The results of grain-size analysis indicate deposition of comparatively coarse-grained silts during the relatively cool and windy conditions of winter and comparatively fine-grained sediments during relatively warm and calm summer months. Pollen analysis demonstrates the significance of long-term storage of pollen grains and fern spores in the estuarine waterbody, superimposed on which seasonal variations in pollen inputs from local and regional vegetation remain detectable. Copyright (C) 2007 John Wiley & Sons, Ltd.

Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.

Assembling spatially explicit landscape models of pollen and spore dispersal by wind for risk assessment

Models of windblown pollen or spore movement are required to predict gene flow from genetically modified (GM) crops and the spread of fungal diseases. We suggest a simple form for a function describing the distance moved by a pollen grain or fungal spore, for use in generic models of dispersal. The function has power-law behaviour over sub-continental distances. We show that air-borne dispersal of rapeseed pollen in two experiments was inconsistent with an exponential model, but was fitted by power-law models, implying a large contribution from distant fields to the catches observed. After allowance for this 'background' by applying Fourier transforms to deconvolve the mixture of distant and local sources, the data were best fit by power-laws with exponents between 1.5 and 2. We also demonstrate that for a simple model of area sources, the median dispersal distance is a function of field radius and that measurement from the source edge can be misleading. Using an inverse-square dispersal distribution deduced from the experimental data and the distribution of rapeseed fields deduced by remote sensing, we successfully predict observed rapeseed pollen density in the city centres of Derby and Leicester (UK).

Updated empirical model of genetically-modified maize grain production practices to achieve European Union labeling thresholds

An updated empirical approach is proposed for specifying coexistence requirements for genetically modified (GM) maize (Zea mays L.) production to ensure compliance with the 0.9% labeling threshold for food and feed in the European Union. The model improves on a previously published (Gustafson et al., 2006) empirical model by adding recent data sources to supplement the original database and including the following additional cases: (i) more than one GM maize source field adjacent to the conventional or organic field, (ii) the possibility of so-called “stacked” varieties with more than one GM trait, and (iii) lower pollen shed in the non-GM receptor field. These additional factors lead to the possibility for somewhat wider combinations of isolation distance and border rows than required in the original version of the empirical model. For instance, in the very conservative case of a 1-ha square non-GM maize field surrounded on all four sides by homozygous GM maize with 12 m isolation (the effective isolation distance for a single GM field), non-GM border rows of 12 m are required to be 95% confident of gene flow less than 0.9% in the non-GM field (with adventitious presence of 0.3%). Stacked traits of higher GM mass fraction and receptor fields of lower pollen shed would require a greater number of border rows to comply with the 0.9% threshold, and an updated extension to the model is provided to quantify these effects.

Resilience of rice (Oryzaspp.) pollen germination and tube growth to temperature stress

Resilience of rice cropping systems to potential global climate change will partly depend on temperature tolerance of pollen germination (PG) and tube growth (PTG). Germination of pollen of high temperature susceptible Oryza glaberrima Steud. (cv. CG14) and O. sativa L. ssp. indica (cv. IR64) and high temperature tolerant O. sativa ssp. aus (cv. N22), was assessed on a 5.6-45.4°C temperature gradient system. Mean maximum PG was 85% at 27°C with 1488 μm PTG at 25°C. The hypothesis that in each pollen grain, minimum temperature requirements (Tn) and maximum temperature limits (Tx) for germination operate independently was accepted by comparing multiplicative and subtractive probability models. The maximum temperature limit for PG in 50% of grains (Tx(50)) was lowest (29.8°C) in IR64 compared with CG14 (34.3°C) and N22 (35.6°C). Standard deviation (sx) of Tx was also low in IR64 (2.3°C) suggesting that the mechanism of IR64's susceptibility to high temperatures may relate to PG. Optimum germination temperatures and thermal times for 1mm PTG were not linked to tolerating high temperatures at anthesis. However, the parameters Tx(50) and sx in the germination model define new pragmatic criteria for successful and resilient PG, preferable to the more traditional cardinal (maximum and minimum) temperatures.

Protein requirements in larvae and adults of Scaptotrigona postica (Hymenoptera: Apidia, Meliponinae): Midgut proteolytic activity and pollen digestion

The number and degree of digestion of pollen grains in the midgut and rectum, the midgut proteolytic activity and the time of pollen grain passage through the digestive tract in the stingless bee Scaptotrigona postica (Latreille) have been analyzed. The results show similar protein requirements among larvae, nurse bees and queens, as well as between forager bees and old males, but these requirements are higher in individuals from the former groups than in those from the latter. Although protein requirements have been demonstrated to vary according to a bee's activity in the colony, they are similar among bees from different castes or sexes. These changes in feeding behavior are related to the bee's function and to less competition for nourishment among individuals of the colony. It is also noted that pollen grains took between 6 and 28 h to pass through the digestive tract. Pollen grains are irregularly accumulated in the various regions of the midgut, which may reflect functional differentiation throughout the midgut. © 2001 Elsevier B.V.

Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)

(Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)). The objective of this work was to characterize the viability, production and morphology of pollen for different species in the genus Manihot. Floral buds from Manihot accessions were collected from two germplasm banks at Embrapa Cassava & Fruits. The viability of the pollen was assessed via colorimetric, in vitro and in vivo assays. The diameter of the pollen grains was determined by measuring the transversal length of the grain. The experimental design was entirely randomized. Studies on pollen ultrastructure were performed via scanning electron microscopy. Pollen viability was high in the colorimetric tests and intermediate in vivo tests; there was no germination in the in vitro tests. The average production for all accessions was 1,253 pollen grains per floral bud. The size of the pollen grains varied from 132 to 163 pm in the wild accessions, and 129 to 146 pm in the cultivated accessions. The pollen grains for all accessions were very large, apolar, spherical as well as inaperturate, with an exine ornamented with pila organized in a Croton pattern. The wild accessions, in general, produced more and larger pollen grains compared with the cultivated accessions.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

Morphological differentiation of Betula (birch) pollen in northwest North America and its palaeoecological application

Lake sediments from arcto-boreal regions commonly contain abundant Betula pollen. However, palaeoenvironmental interpretations of Betula pollen are often ambiguous because of the lack of reliable morphological features to distinguish among ecologically distinct Betula species in western North America. We measured the grain diameters and pore depths of pollen from three tree-birch species (B. papyrifera, B. kenaica and B. neoalaskana) and two shrub-birch species (B. glandulosa and B. nana), and calculated the ratio of grain diameter to pore depth (D/P ratio). No statistical difference exists in all three parameters between the shrub-birch species or between two of the tree-birch species (B. kenaica and B. papyrifera), and B. neoalaskana is intermediate between the shrub-birch and the other two tree-birch species. However, mean pore depth is significantly larger for the tree species than for the shrub species. In contrast, mean grain diameter cannot distinguish tree and shrub species. Mean D/P ratio separates tree and shrub species less clearly than pore depth, but this ratio can be used for verification. The threshold for distinguishing pollen of tree versus shrub birch lies at 2.55 μm and 8.30 for pore depth and D/P ratio, respectively. We'applied these thresholds to the analysis of Betula pollen in an Alaskan lake-sediment core spanning the past 800 years. Results show that shrub birch increased markedly at the expense of tree birch during the‘Little Ice Age’; this patten is not discernible in the profile of total birch pollen.

Minimum count sums for charcoalconcentration estimates in pollen slides: accuracy and potential errors

Charcoal particles in pollen slides are often abundant, and thus analysts are faced with the problem of setting the minimum counting sum as small as possible in order to save time. We analysed the reliability of charcoal-concentration estimates based on different counting sums, using simulated low-to high-count samples. Bootstrap simulations indicate that the variability of inferred charcoal concentrations increases progressively with decreasing sums. Below 200 items (i.e., the sum of charcoal particles and exotic marker grains), reconstructed fire incidence is either too high or too low. Statistical comparisons show that the means of bootstrap simulations stabilize after 200 counts. Moreover, a count of 200-300 items is sufficient to produce a charcoal-concentration estimate with less than+5% error if compared with high-count samples of 1000 items for charcoal/marker grain ratios 0.1-0.91. If, however, this ratio is extremely high or low (> 0.91 or < 0.1) and if such samples are frequent, we suggest that marker grains are reduced or added prior to new sample processing.

Pollen record and age determination of a sediment profile from Frengstadsetra, Norway

Innerdalen was once a mountain valley (ca. 780 m a.s.l.) with birch forests, bogs and several summer farms. Today it is a 6.5 km**2 artifical lake. In 1980 and 1981 archaeological and palynological investigations were carried out due to the hydroelectric power plans. Radiocarbon dated pollen diagrams from 9 different localities in Innerdalen provide information on a mountain environment which has been exploited to varying degrees by human groups for thousands of years. In the Birch Zone, ca. 9500-8500 years B.P., the deglaciated surface is vegetated by the normal sequence of pioneering species, first show-bed communities, then shrub/dwarf-shrub communities, and finally a birch forest community. In the Pine Zone, ca. 8500-7500 years B.P., the mixed Birch-Pine forest which prevailed at the end of the Birch Zone is replaced by a dense pine forest. The tree limit was higher than it is today. In the Alder Zone, ca. 7500-4000 years B.P., the newly arrived alder gradually succeeded pine, particularily on good soils. This alder forest has a modem analog in the pre-alpine gray alder forests in Norway. In the last part of the Alder Zone, ca. 6000-4000 years B.P., elm and hazel are nominally present on particularily rich soils, marking the edaphic and climatic optimum in Innerdalen. During this time the first evidence of human impact on the vegetation is apparent in the pollen diagrams. At both Sætersetra in the south of the valley and Liabekken in the north, forest clearance and the development of grazed grass meadows is documented, and human impact continues until the present. The Herb Zone, ca. 4000 years B.P. to 1600 A.D., is characterized by the rapid decline of alder. The forest is increasingly open, and bog formation is initiated. The sub-alpine belt of birch forest is established, probably due to the shift to a cooler, moister climate. Human activity can also have influenced the vegetational changes, although at 4 of the localities human activity also is first apparent after the alder decline. Some localities show measurably less human impact on the vegetation ca. 2600-2000 years B.P. Grazing intensity increases ca. 2000 years B.P. At the end of the Herb Zone rye and barley pollen is registered at Sætersetra and Flonan, indicating contact between the grazing activities of Innerdal and grain cultivation activities outside the valley. The Spruce Zone, ca. 1600 A.D. to the present, does not begin synchronously since the presence of long-distance transported spruce pollen at a locality is entirely dependent on the density of the vegetation ie. degree of human impact. The youngest spruce rise is ca. 1500 A.D. at Røstvangen, when summerfarming is initiated. Summerfarming activities in Innerdal produce an increasingly open landscape. Rye and barley pollen at several localities may indicate limited local cultivation, but is more likely long-distance transport via humans and domesticated animals from cultivated areas outside Innerdalen.