Similar taxonomic richness but different communities of ectomycorrhizas in native forests and non-native plantation forests

This investigation sought to examine if there was a difference between the ectomycorrhizal (ECM) communities in plots of native oak and introduced Scots pine and Sitka spruce forest. The ECM communities in four plots of each forest type were described, from five soil cores collected in each plot, by morphotyping, internal transcribed spacer (ITS)-restriction fragment length polymorphism matching of mycorrhizas and sporocarps and ITS sequencing. Fifty-one distinct taxa were distinguished; 25 were identified to species level, 11 to genus and 15 remained unidentified. Seventy-one ECM species were recorded as sporocarps from the forest plots; most (43 species) were found in the Sitka spruce plots. The below-ground ECM communities of the different forest types did not differ significantly with respect to species richness of taxa on roots, but differed in species composition. Multivariate analysis produced a clear separation of the communities of the different forest types using below-ground data, but the above-ground sporocarp data did not separate the forest types. Moreover, results of a Mantel test found no relationship between the above- and below-ground similarity matrices. The oak plots had the most distinctive ECM community, with Laccaria amethystina and Elaphomyces granulatus being frequent. The Sitka spruce plots showed the lowest intra-forest type similarity and were often dominated by "nursery type" ectomycorrhizas. There was only 10% similarity between the above- and below-ground ECM species in these plots, different colonisation methods of ectomycorrhizal taxa and insufficient below-ground sampling being possible reasons for this disparity. Our results indicate that plantations of non-native Sitka spruce can support similar levels of ECM diversity as native forests.     

Spatial structure and diversity of woody plants and ectomycorrhizal fungus sporocarps in a natural subtropical forest

Spatial patterns of ectomycorrhizal fungi, ectomycorrhizal plants, and non-ectomycorrhizal plants were investigated in a natural subtropical forest using second-order analysis. The results of spatial pattern analysis showed that the degree of clumping of woody plants and ectomycorrhizal sporocarps were correlated. There was a significantly positive correlation of relative aggregation indices between ectomycorrhizal fungi and both non-ectomycorrhizal trees and ectomycorrhizal saplings. Correlations between percentage of ectomycorrhizal trees and sporocarp occurrence of ectomycorrhizal fungi and between diversities of woody plants and ectomycorrhizal fungi were distance-dependent or scale-related. A significantly high percentage of ectomycorrhizal trees was found only at relatively short distance from ectomycorrhizal fungal sporocarps, and significantly positive correlation of the diversity between woody plants and ectomycorrhizal fungi was found only at relative long distance, which implied that ectomycorrhizal sporocarps prefer ectomycorrhizal-tree-dominant micro-sites at near distances and at relatively large scales, diverse ectomycorrhizal sporocarps could be found in woodlands with high diversity of woody plants. Important factors affecting the spatial distribution, occurrence, and diversity of ectomycorrhizal fungi include spatial pattern of ectomycorrhizal plants and non-ectomycorrhizal plants, percentage of ectomycorrhizal plants, and plant diversity in a natural forest.     

四川都江堰亚热带针阔混交林中主要晚生型外生菌根真菌类群的空间分布

晚生型外生菌根真菌通常出现在森林演替的后期,是成熟林中的优势外生菌根真菌类群.对四川都江堰一片亚热带针阔混交林中的菌根真菌地上群落进行调查,并应用二元逻辑回归分析对晚生型外生菌根真菌的主要类群,即鹅膏菌科、牛肝菌科和红菇科,与周围(5 m×5 m样方)树种组成的关系进行研究.还应用次级变量分析方法对主要外生菌根真菌类群的空间格局进行了分析.结果表明,非外生菌根树种及某些外生菌根树种对特定类群菌根真菌子实体的出现有抑制作用,而不同类群外生菌根真菌在克隆生长上的差异并不是子实体空间分布的决定因素.我们认为,当研究自然林中外生菌根子实体的空间分布时,除了宿主植物的分布,也应考虑非宿主植物的分布以及菌根真菌相互作用的影响.     

四川都江堰亚热带针阔混交林中主要晚生型外生菌根真菌类群的空间分布

晚生型外生菌根真菌通常出现在森林演替的后期,是成熟林中的优势外生菌根真菌类群。对四川都江堰一片亚热带针阔混交林中的菌根真菌地上群落进行调查,并应用二元逻辑回归分析对晚生型外生菌根真菌的主要类群,即鹅膏菌科、牛肝菌科和红菇科,与周围(5m×5m样方)树种组成的关系进行研究。还应用次级变量分析方法对主要外生菌根真菌类群的空间格局进行了分析。结果表明,非外生菌根树种及某些外生菌根树种对特定类群菌根真菌子实体的出现有抑制作用,而不同类群外生菌根真菌在克隆生长上的差异并不是子实体空间分布的决定因素。我们认为,当研究自然林中外生菌根子实体的空间分布时,除了宿主植物的分布,也应考虑非宿主植物的分布以及菌根真菌相互作用的影响。     

Patterns and drivers of lichen species composition in a NW-European lowland deciduous woodland complex

We surveyed lichens in an extensive area of lowland deciduous oak and beech dominated forest under wood pasture management within the New Forest National Park, southern England. This provided the basis for an investigation of factors affecting the species density and composition of epiphytic lichen communities. Fifteen 1 hectare plots were established in the same sites as a parallel invertebrate survey, of which nine were in old growth forest and six in oak plantations of between c. 150 and 300 years old. In each plot 12 trees were identified for sampling and lichens were sampled on four aspects of each trunk. Results at the plot level showed that species density was significantly higher in the old growth woodland plots from those in plantations. Plot age had a significant effect on species on oak but this was not significant for lichen communities on beech. The species density of lichens associated with Trentepohliaceae photobionts on oaks showed a linear positive relationship with increasing plot age while the species density of species with other coccal green Chlorophyta decreased with age of the plot. A major part of the lichenised fungi with Trentepohliaceae photobionts includes indicator and notable species used in conservation evaluation and this component is most affected by fragmentation and isolation of forest sites. The results emphasise the importance of long term connectivity and ecological continuity in this extensive mosaic of lowland deciduous forest.     

Forest microsite effects on community composition of ectomycorrhizal fungi on seedlings of Picea abies and Betula pendula

Niche differentiation in soil horizons, host species and natural nutrient gradients contribute to the high diversity of ectomycorrhizal fungi in boreal forests. This study aims at documenting the diversity and community composition of ectomycorrhizal fungi of Norway spruce ( Picea abies ) and silver birch ( Betula pendula ) seedlings in five most abundant microsites in three Estonian old-growth forests. Undisturbed forest floor, windthrow mounds and pits harboured more species than brown- and white-rotted wood. Several species of ectomycorrhizal fungi were differentially represented on either hosts, microsites and sites. Generally, the most frequent species in dead wood were also common in forest floor soil. Ordination analyses suggested that decay type determined the composition of EcM fungal community in dead wood. Root connections with in-growing mature tree roots from below affected the occurrence of certain fungal species on seedling roots systems in dead wood. This study demonstrates that ectomycorrhizal fungi differentially establish in certain forest microsites that is attributable to their dispersal and competitive abilities. Elevated microsites, especially decayed wood, act as seed beds for both ectomycorrhizal forest trees and fungi, thus affecting the succession of boreal forest ecosystems.     

Ectomycorrhizal and wood-decay macromycete communities along development stages of managed Pinus patula stands in Southwest Mexico

This study used fruit body surveys to evaluate the diversity and distribution of ectomycorrhizal and wood-decay macromycetes along developing stages of stands artificially reforested with Pinus patula in Southern Mexico. Three stand ages were selected: 1, 11, and 60 y old. Macromycetes were collected from June to October 2016 in 0.1 ha plots per stand age. A total of 106 taxa were recorded, 63 ectomycorrhizal and 43 wood-decay fungi. The highest diversity of ectomycorrhizal fungi was in 11-y stands, and 11- and 60-y stands had similar species composition. Wood-decay fungi showed a trend towards higher diversity in the 1- and 11-y stands, and species composition in these sites corresponded. Patterns of diversity and distribution were mainly determined by factors related to substratum availability and vegetation structure. Knowledge about the influence of artificially reforested stands on macrofungal communities is relevant to improve management plans and achieve a more sustainable use of forest timber and non-timber products.     

Influence of host species on ectomycorrhizal communities associated with two co-occurring oaks (Quercus spp.) in a tropical cloud forest

Interactions between host tree species and ectomycorrhizal fungi are important in structuring ectomycorrhizal communities, but there are only a few studies on host influence of congeneric trees. We investigated ectomycorrhizal community assemblages on roots of deciduous Quercus crassifolia and evergreen Quercus laurina in a tropical montane cloud forest, one of the most endangered tropical forest ecosystems. Ectomycorrhizal fungi were identified by sequencing internal transcribed spacer and partial 28S rRNA gene. We sampled 80 soil cores and documented high ectomycorrhizal diversity with a total of 154 taxa. Canonical correspondence analysis indicated that oak host was significant in explaining some of the variation in ectomycorrhizal communities, despite the fact that the two Quercus species belong to the same red oak lineage (section Lobatae ). A Tuber species, found in 23% of the soil cores, was the most frequent taxon. Similar to oak-dominated ectomycorrhizal communities in temperate forests, Thelephoraceae, Russulaceae and Sebacinales were diverse and dominant.     

Ectomycorrhizal fungi are shared between seedlings and adults in a monodominant Gilbertiodendron dewevrei rain forest in Cameroon

Ectomycorrhizal networks may facilitate the establishment and survival of seedlings regenerating under the canopies of tropical forests and are often invoked as a potential contributor to monodominance. We identified ectomycorrhizal fungi in a monodominant Gilbertiodendron dewevrei (Fabaceae) rain forest in Cameroon, using sporocarps and ectomycorrhizae of three age categories (seedlings, intermediate trees, and large trees) and tentatively revealed nutrient transfer through ectomycorrhizal networks by measuring spontaneous isotopic (¹³C and ¹⁵N) abundances in seedlings. Sporocarp surveys revealed fewer ectomycorrhizal fungal taxa (59 species from 1030 sporocarps) than molecular barcoding of ectomycorrhizal roots (75 operational taxonomic units from 828 ectomycorrhizae). Our observations suggest that ectomycorrhizal fungal diversity is similar to that in other mixed tropical forests and provide the first report of the Tuber‐Helvella lineage in a tropical forest. Despite some differences, all age categories of G. dewevrei had overlapping ectomycorrhizal fungal communities, with families belonging to Thelephoraceae, Russulaceae, Sebacinaceae, Boletaceae, and Clavulinaceae. Of the 49 operational taxonomic units shared by the three age categories (65.3% of the ectomycorrhizal fungal community), 19 were the most abundant on root tips of all categories (38.7% of the shared taxa), supporting the likelihood of ectomycorrhizal networks. However, we obtained no evidence for nutrient transfer from trees to seedlings. We discuss the composition of the ectomycorrhizal fungal community among the G. dewevrei age categories and the possible role of common ectomycorrhizal networks in this rain forest.     

Soil burrowing Muscina angustifrons (Diptera: Muscidae) larvae excrete spores capable of forming mycorrhizae underground

Muscina angustifrons (Diptera: Muscidae) is a mycophagous species that exploits a variety of fungi, including ectomycorrhizal fungi. Larvae of this species have been shown to feed on sporocarps (including spores), and full-grown larvae leave sporocarps and pupate 0–6?cm below the soil surface. In this study, we examined whether M. angustifrons larvae are capable of transporting ectomycorrhizal fungal spores and enhancing ectomycorrhiza growth on host-plant roots. Full-grown larvae were found to move horizontally 10–20?cm from their feeding sites and burrow underground. These wandering larvae retained ectomycorrhizal fungal spores in their intestines, which were excreted following relocation to underground pupation sites. Excreted spores retained germination and infection capacities to form ectomycorrhiza on host-plant roots. In the infection experiments, ectomycorrhizal fungal spores applied in the vicinity of underground host-plant roots were more effective in forming ectomycorrhiza than those applied to the ground surface, suggesting that belowground transportation of spores by M. angustifrons larvae could enhance ectomycorrhizal formation. These results suggested that M. angustifrons larvae act as a short-distance spore transporter of ectomycorrhizal fungi.     

Ectomycorrhizal fungal sporocarp diversity and discovery of new taxa in Dicymbe monodominant forests of the Guiana Shield

Ectomycorrhizal (ECM) fungi historically were considered poorly represented in Neotropical forests but in the central Guiana Shield substantial areas are dominated by leguminous ECM trees. In the Upper Potaro Basin of Western Guyana, ECM fungi were sampled for 7?years during the rainy seasons of 2000–2008 in three 1-ha plots in primary monodominant forests of the ECM canopy tree Dicymbe corymbosa (Fabaceae subfam. Caesalpinioideae). Over the plot sampling period sporocarps of 126 species of putative or confirmed ECM fungi were recovered. These taxa represented 13 families and 25 genera of primarily Agaricomycetes, but also Ascomycota (Elaphomycetaceae), the majority of which are new to science. Russulaceae contained the most species (20 Russula; 9 Lactarius), followed by Boletaceae (8 genera, 25 spp.), Clavulinaceae (17 Clavulina), and Amanitaceae (16 Amanita). An additional 46 species of ECM fungi were collected in forests of the Upper Potaro Basin outside the study plots between 2000 and 2010, bringing the regional number of ECM species known from sporocarps to 172. This is the first long-term ECM macrofungal dataset from an ECM-dominated Neotropical forest, and sporocarp diversity is comparable to that recorded for ECM-diverse temperate and boreal forests. While a species accumulation curve indicated that ECM sporocarp diversity was not fully recovered inside of the plots,?~80% of the total species were recovered in the first year. Sequence data from ECM roots have confirmed the ECM status of 56 taxa represented by corresponding sporocarp data. However,?>50% of ECM fungal species from roots remain undiscovered as sporocarps, leading to a conservative estimate of?>?250 ECM species at the Potaro site. Dicymbe forests in Guyana are a hotspot for ECM fungal diversity in the Neotropics.     

Do lianas shape ant communities in an early successional tropical forest?

Almost half of lowland tropical forests are at various stages of regeneration following deforestation or fragmentation. Changes in tree communities along successional gradients have predictable bottom‐up effects on consumers. Liana (woody vine) assemblages also change with succession, but their effects on animal succession remain unexplored. Here we used a large‐scale liana removal experiment across a forest successional chronosequence (7–31 years) to determine the importance of lianas to ant community structure. We conducted 1,088 surveys of ants foraging on and living in trees using tree trunk baiting and hand‐collecting techniques at 34 paired forest plots, half of which had all lianas removed. Ant species composition, β‐diversity, and species richness were not affected by liana removal; however, ant species co‐occurrence (the coexistence of two or more species in a single tree) was more frequent in control plots, where lianas were present, versus removal plots. Forest stand age had a larger effect on ant community structure than the presence of lianas. Mean ant species richness in a forest plot increased by ca. 10% with increasing forest age across the 31‐year chronosequence. Ant surveys from forest >20 years old included more canopy specialists and fewer ground‐nesting ant species versus those from forests <20 years old. Consequently, lianas had a minimal effect on arboreal ant communities in this early successional forest, where rapidly changing tree community structure was more important to ant species richness and composition.     

Age class influence on the yield of edible fungi in a managed Mediterranean forest

Lack of information and difficulty in predicting wild edible sporocarp yields is blocking its integration in forest management. In the Mediterranean area, this nontimber forest product has increased its market value, consumption demand, and interest over the last decade. In this work, sampling year and stand age effects are analyzed in order to advance knowledge of edible fungi community structure, dynamics, and production. Weekly autumnal sporocarp monitoring was performed from 1997 to 2011 in a Pinus pinaster managed forest in central Spain. After applying a random stand age-stratified survey, 21 plots of 150 m² have been set with three per stand age class. The forest age classes have been defined as follows: 0–10 years, mixture of parent and regenerated trees, 11–20, 21–40, 41–60, 61–90, and over 90 years. A total of 153 species belonging to 56 genera were recorded, 55 of which are edible. The production of edible sporocarps was 19.8 kg ha^?1, representing 31 % of total production. Sporocarp production presents a sharp interannual variability with autumns 62 times more productive than others. The most abundant edible species in terms of fresh weight per hectare has been Lactarius deliciosus with 7.0 kg ha^?1. Edible fungi yields registered a significant decline in 10 years following regenerative cutting. The presence of parent trees significantly increases production with regard to the first class. The highest production of edible species occurs in the middle age, 41–60 years, and in the following classes, a decrease is produced. L. deliciosus production registered differences with age, manifesting in a high yield in young stands (11–20 years) and significant recovery in woodlands near to the cutting.     

Changes in two Minnesota forests during 14 years following catastrophic windthrow

Abstract. We measured tree damage and mortality following a catastrophic windthrow in permanent plots in an oak forest and a pine forest in central Minnesota. We monitored changes in forest structure and composition over the next 14 years. Prior to the storm, the oak forest was dominated by Quercus ellipsoidalis, and the pine forest by Pinus strobus. The immediate impacts of the storm were to differentially damage and kill large, early‐successional hardwoods and pines. Subsequent recovery was characterized by the growth of late‐successional hardwoods. In both forests the disturbance acted to accelerate succession. Ordination of tree species composition confirmed the trend of accelerated succession, and suggested a convergence of composition between the two forests.     

Assemblages of wood-inhabiting fungi along the gradients of succession and naturalness in boreal pine-dominated forests in Fennoscandia

In boreal forests, the level of naturalness and the stage of succession explain most of the variation in forest structure within a particular forest type. Thus, these two factors should also have a major effect on species assemblages in forests, at least on species groups associated with wood. The present study is the first attempt to analyze empirically the simultaneous effects of forest succession and naturalness on wood-inhabiting fungi, a taxonomic group of special ecological importance. The study area was situated in eastern Finland, middle boreal zone. A total of 41 study plots were established in Pinus sylvestris forests representing three levels of forest naturalness: natural, seminatural and intensively managed forests. Five stages of succession were distinguished according to the age of the dominating tree layer (<10, 40, 70, 110, and >150 yr old), except in managed forests where only four stages were available. A total of 5328 records of 195 species of fungi were made. The first, open stage of succession was clearly the most species-rich period of succession in all levels of forest naturalness. In natural and seminatural forests, the first stage of succession was also very distinctive in its fungal composition, and thus of special value in protecting biodiversity in boreal forests. In the succession following the first stage, the level of naturalness had more effect on assemblages of fungi than did the stage of succession. Intensive forest management affects threatened species particularly. In conclusion, natural young stages of succession should also be included in the network of boreal forest reserves.     

Allocasuarina tree hosts determine the spatial distribution of hypogeous fungal sporocarps in three tropical Australian sclerophyll forests

Across three tropical Australian sclerophyll forest types, site-specific environmental variables could explain the distribution of both quantity (abundance and biomass) and richness (genus and species) of hypogeous fungi sporocarps. Quantity was significantly higher in the Allocasuarina forest sites that had high soil nitrogen but low phosphorous. Three genera of hypogeous fungi were found exclusively in Allocasuarina forest sites including Gummiglobus, Labyrinthomyces and Octaviania, as were some species of Castoreum, Chondrogaster, Endogone, Hysterangium and Russula. However, the forest types did not all group according to site-scale variables and subsequently the taxonomic assemblages were not significantly different between the three forest types. At site scale, significant negative relationships were found between phosphorous concentration and the quantity of hypogeous fungi sporocarps. Using a multivariate information theoretic approach, there were other more plausible models to explain the patterns of sporocarp richness. Both the mean number of fungal genera and species increased with the number of Allocasuarina stems, at the same time decreasing with the number of Eucalyptus stems. The optimal conditions for promoting hypogeous fungi sporocarp quantity and sporocarp richness appear to be related to the presence and abundance of Allocasuarina (Casuarinaceae) host trees. Allocasuarina tree species may have a higher host receptivity for ectomycorrhizal hypogeous fungi species that provide an important food resource for Australian mycophagous animals.     

Endophytes dominate fungal communities in six-year-old veteranisation wounds in living oak trunks

Old trees are rare in the landscape, as are many of their associated species. Veteranisation is a method by which attempts are made to create microhabitats, otherwise found only in old trees, in younger trees at an earlier stage than would occur naturally. Here, we analysed the early fungal succession in 6 y-old veteranisation wounds in ca. 100 y old living oak trunks by DNA-barcoding of the wood at eight sites in Sweden and Norway. We hypothesised basidiomycetes would be most abundant, and exposed sapwood and heartwood would select for different communities. We identified 686 fungal taxa, mainly ascomycetes, with a large overlap in species composition and surprisingly similar species richness, i.e. 325 vs. 308–360, between intact and different types of damaged wood, respectively. Endophytes continued to be present and common in damaged wood. The results demonstrate that damage to sapwood and heartwood partly select for different fungi and that 6 y is too early to evaluate if veteranisation can positively favour fungi of conservation interest.     

Avalanches creating high structural and floristic diversity in mountain mixed forests in the Alps

On January 18, 1986 a snow avalanche fell down from the steep slope of Mount Watzmann, Berchtesgaden National Park, Germany, into a mountain mixed forest reaching an elevation of only 610 m asl. The trees were bent to the ground, but they survived, forming a dense canopy pressed to the ground. Although on the landscape scale the avalanche marked a strong visual impact we hypothesized that the species composition of the forest ground layer would not change significantly. To test this we established a set of permanent plots. In the following years new avalanches over and adjacent to the former avalanche track occurred. The established permanent plots allow us to evaluate a second hypothesis, namely that not only structural diversity has been enhanced by the avalanches, but also a sequence of different succession pathways initiated which all together will keep diversity at a high level in the affected area. Our analysis of forest patches which were affected only by the first avalanche shows that, although a strong change in forest structure has taken place, the species composition here remained to a large extent unchanged. This suggests that beneath the dense crown canopy built up by the bent trees the microclimate shows little change, mechanical soil disturbances are rare, and therefore species of open land have no chance to establish. In contrast, if the avalanche uproots and kills the trees a clear change in species composition is the result. On our permanent plots we were able to identify eight different succession pathways initiated by the different disturbance events, causing at various points of time a set of different development phases of the forest ecosystem on the avalanche track.     

Impacts of anthropogenic disturbances on forest succession in the mid-montane forests of Central Himalaya

We studied the influence of anthropogenic drivers on the distribution and regeneration of tree species in vegetation at different stages of succession from grasslands to oak forests in mid-montane Central Himalaya. We found fire, grazing, and lopping as the main factors hindering a progressive successional regime towards a late-successional oak community. Succession was studied in five vegetation formations (grasslands, pine, pine–oak, open oak, and dense oak), with similar site conditions, representing a theoretical successional sequence from early- to late-successional stages. A structured survey with uniform distribution of sampling plots in the five selected vegetation formations was conducted to gather information abut the vegetation communities. Early-successional grasslands and pine forests were found to harbour high densities of pine and oak seedling and sapling regeneration. However, recurring fires and chronic unsustainable levels of grazing in these vegetation formations obstructed progressive succession by eliminating regenerating seedling and saplings from the forest understorey. Similarly, in intermediate- and late-successional stages (including pine–oak, open oak, and dense oak), overexploitation of existing oaks trees via lopping and grazing of regenerating oak seedlings and saplings hampered oak regeneration and development. The possibility to convert pine forests into oak as well as the conservation of existing oak forests through controlled grazing and lopping are management options that can contribute to an enhanced functioning of forest ecosystems in the study area. We conclude that with strategic management that restricts the current anthropogenic disturbances, the extent of oak forest in the study area can be increased.     

Changes in the community of ectomycorrhizal fungi and increased fine root number under adult beech trees chronically fumigated with double ambient ozone concentration

Forest soils are an important but under-studied part of forest ecosystems. The effects of O(3) on below-ground processes in a mature forest have only received limited attention so far. In our study, we have analysed the community of ectomycorrhizal fungi and beech fine root dynamics over two growing seasons (2003 - 2004) in a 70-year old mixed spruce-beech forest stand, in which two groups of five adult beech trees were either fumigated by 2 x ambient ozone concentration or used as control. The main difference between previous studies and our approach was that previous studies were performed on seedlings in pot experiments or in closed or open top chambers, and not IN SITU, in a mature forest stand. Although beech is a relatively unresponsive species to tropospheric O(3), we found a pronounced effect of 2 x O(3) on the number of vital ectomycorrhizal root tips and non-turgescent fine roots. Both categories of roots were significantly increased when compared to controls in two consecutive years at each sampling event. The number of types of ectomycorrhizae and species richness increased in 2004, but not in the extremely dry year 2003. We hypothesised that the observed changes might be an expression of a transitional state in below-ground succession of niches caused by an O(3) induced effect on carbon allocation to roots and the rhizosphere. We have detected changes in ectomycorrhizal species level, however Shannon-Weavers species diversity index and percentage of types of ectomycorrhizae did not change significantly in any sampling year thus indicating our results cannot be unequivocally explained by summer drought in year 2003 or by O(3) exposure alone.