Pollen Size Research Articles

Pollen morphology of the three subgenera of Alnus

The three subgenera of alder (Alnusroman Clethropsis and Alnobetula) are taxonomically distinctive, but how does their pollen match up with their gross morphology? This study links pollen characters with the taxonomic divisions of the genus alder based on nrDNA ITS sequence data. The value of the study is in the breadth of the taxonomic coverage of the three subgenera, as the samples per species only range from 1 to 6. From 29 Alnus species, the authors studied pollen from 59 modern reference collections (prepared by acetolysis) and recorded the number and type of apertures (pores), thickness of the arci, pollen size and polar arci. Tallies of the number of pores on the pollen grains in a reference collection typically have a range of ± 1 or 2. Exceptions are presumed hybrid collections in Alnus rugosa with a range of ± 5, which have malformed pollen cells suggesting infertility. The presumed hybrids show a greater range of both pore number and size of pollen grains. The dominant pore number is a key feature that separates the subgenus Alnobetula (= Alnaster) from the other two subgenera by consistently having predominantly 5-, 6- or 7-pored pollen grains, while all other Alnus taxa sampled have pollen with predominantly 4- or 5-pored pollen. The dominant pore number is a consistent feature within the subgenus Alnobetula (10 species), but is inconsistent in 3 out of 16 species of the subgenus Alnus and within the subgenus Clethropsis (3 species). Thickenings of the pore lip, aspidate (protruding) and vestibulate pores are important features of Alnus pollen on a species level. Alnus-like pollen morphology occurs occasionally in certain other betulaceous genera (Betula and Carpinus). Differences in character or thickness of arci (band-like thickenings on the pollen wall between pores) tend to be somewhat consistent within a species and vary within subgenera. Two Alnobetula species in the Alnus viridis complex (Alnus crispa and Alnus sinuata) have arci that are weakly developed or appear as simple folds; walls are fragile and pores are typically less aspidate. Pollen sizes in different species overlap greatly, may be partly influenced by processing methods and do not seem to be generally helpful in characterizing species. Circular doughnut-shaped arci thickenings on the polar area of pollen grains are a common feature (16–40%) in two closely related East Asian taxa of subgenus Alnobetula with large pollen (‘Clade A’: Alnus firma and Alnus sieboldii). These ‘Clade A’ features have been reported from Miocene collections of Alnus pollen from Alaska, and support megafossil evidence in determining the biogeography of this group along the Pacific Rim in the Neogene. The Miocene appearance of subgenus Alnobetula megafossils in both Alaska and Japan can only have occurred via a Bering Land Bridge. This relation is now confirmed by Reinink-Smith's Miocene ‘Clade-A-type’ pollen data of Alaska. These taxa had Pacific Rim distributions, similar fruiting carposamaras and now correspond to a DNA ITS subgroup within Alnobetula. Depending on the geographic location with limited number of local alder species, certain Holocene identifications are possible based on pore structure and arci thickness, e.g. the Alnus crispa-type in eastern Canada and the Alnus sinuata-type in the Pacific northwest.

Early Holocene hybridisation between Betula pubescens and B. nana in relation to birch vegetation in Southwest Iceland

The aims of the present research were to gain better insight into the early Holocene vegetation history of Betula in South Iceland and to investigate whether hybridisation between downy tree-birch (Betula pubescens) and dwarf birch (Betula nana) could be discovered in this region, similar to that previously found in North Iceland. A peat monolith spanning the period from about 10.3 to 7.6calkaBP from Eyvík, Southwest Iceland, provided 44 samples for pollen analysis. The samples were dated by known tephra layers, Saksunarvatn and Seydishólar tephras, and two 14C datings. The macrofossil and spore/pollen results showed several progressive phases of vegetation development in the area until birch woodland became established around 9.5calkaBP. The timing of birch colonisation is in good agreement with results from other sites in Iceland. From these Eyvík samples, all morphologically normal Betula pollen grains were size-measured and abnormal pollen grains recorded. Species proportions within samples were estimated statistically on the basis of pollen size. As birch woodland in this area became established rather late, statistical analysis of Betula pollen size was only possible in the uppermost 18 samples, approximately spanning the period between 7.6 and 9.5calkaBP. The frequency distribution of pollen size indicated populations of B. nana predating those of B. pubescens. The proportion of B. pubescens pollen was oscillating but increased with time. In two samples at the top of the peat monolith, more than half of the measured Betula pollen grains belonged to B. pubescens. Evidence of hybridisation based on high frequencies of non-triporate Betula pollen was found in two samples dated at about 8.8 and 7.7calkaBP. Conditions appeared to be comparable to the corresponding hybridisation events previously reported for North Iceland, i.e. in warm periods when downy birch was expanding near dwarf birch habitats.

Chapter 1: An overview of allergens

Most allergens are proteins or glycoproteins that range in molecular weight from 5000 to 100,000 Da, although polysaccharides and low molecular weight substances also may be allergenic. Common allergens include pollens, fungal spores, house-dust mites, and animal epithelial materials but can also include drugs, biological products, and insect venoms. The allergic response is dependent on the route of exposure. If exposure is to an inhaled aeroallergen, the allergic response will be a respiratory reaction in nature. Ingested or injected exposure gives rise to gastrointestinal, cutaneous, or anaphylactic reactions. Size of pollen determines clinical manifestation of allergy. For example, particles between 20 and 60 μm in diameter can be carried in the wind and cause nasal and ocular symptoms (allergic rhinoconjunctivitis). Particles <7 μm can deposit in the airways and cause symptoms of asthma. Animals produce allergens in forms unique to each species. Cat allergen, most importantly Fel d 1, is found mainly in cat saliva, sebaceous glands in the skin, and in urine of male cats. It is buoyant and "sticky," which means it easily remains airborne and may last in a home for up to 6-9 months after the source is removed. Cat allergen adheres to clothes and can be found in public places such as schools. Dog allergen, particularly Can f 1, is present in dander, saliva, urine, and serum. There are allergens specific to dog breeds, but all breeds produce allergenic proteins (even poodles and "hairless" dogs).

Palynological diversity in selected medicinal plant species of Asteraceae (Compositae) from flora of Kaghan Valley

Pollen morphology of seven species, belonging to seven Asteraceae genera from the flora of Kaghan Valley was investigated by light and scanning electron microscopy. The species named Achillea millefolium Linn., Chrysanthemum leucanthemum Linn., Gerbera gossypina (Royle) Beauv., Senecio chrysanthemoides DC. Prodr., Sonchus asper Linn.,Tagetes erecta Linn. and Xanthium strumarium Linn. were studied which showed considerable variation in pollen shape, size, length and number of spines, colpi and sculpturing in light as well as electron microscopy. The maximum pollen size was found inG. gossypina which was 38.3 µm in polar view and 57.75 in µm equatorial view whereas minimum pollen size in polar view that is, 23.65 µm was found in X. strumarium and 19 µm in equatorial view was found in A. millefolium. Psilate pollen sculpturing was observed in G. gossypina however the other species had spines on their pollen. The psilate sculpturing was considered as an advanced feature when compared to echinate sculpturing of pollen which was regarded as the primitive form. It was concluded that this research work has provided new dimensions towards further investigations in the field of palynology. Key words: Palynology, psilate, echinate.

Pollen morphology of the genusPotentilla(Rosaceae) in Iran

The pollen morphology of 28 species of the genus Potentilla (Rosaceae) and four related genera from Iran was examined using light (LM) and scanning electron microscopy (SEM). The pollen grains are radially symmetrical, isopolar to subisopolar, trizonocolpate and colporate. The shape of pollen grains in all the studied taxa varies from prolate-spheroidal to subprolate and prolate. The outline of the pollen grains is elliptical in equatorial view and triangular-circular in polar view. The size of the pollen grains is small to medium. Our results showed two main types of exine sculpturing. Among the studied pollen grain characters, shape and size are helpful evidences for separating different sections, while the exine sculpturing type provides limited taxonomic value.

Palynological study of the Venezuelan species of the genus Hymenocallis (Amaryllidaceae)

Pollen morphology and exine structure of the seven Hymenocallis Salisb. species present in Venezuela were investigated using scanning (SEM) and transmission (TEM) electron microscopy. Pollen grains of all species are monosulcate, heteropolar, with bilateral symmetry, oblate to peroblate, heterobrochate, semitectate-columellate, possess unequal tectal surfaces, and are clavate or baculate when viewed with TEM. They present two equatorial apexes. When observed under SEM their surfaces appear as granulate; however, it is evident from TEM that they are in fact constituted by pila. All the species possess very large pollen (ranging from 64 to 85 μm and from 125 to 155 μm for polar and equatorial axes, respectively), and no relationship was evident between pollen size and chromosome number. All the taxa are also very similar in relation to their pollen outline, ornamentation and internal structure, thus indicating that the genus is accurately delimited from the palynological point of view and that discrete characteristics can only be used in some cases to delimit individual species.

Morphological, karyological and taxonomic remarks on Ferulago nodosa (L.) Boiss. (Apiaceae)

Western (Sicilian) and eastern (Balcan-aegean) populations of the Balcan-tyrrhenian Ferulago nodosa are reported to be different in some morphological characters, and were considered different species by some authors in XIX century. In this work, fruit and pollen morphologies have been compared in Sicilian and Cretan plants; also, the chromosome number of Sicilian plants has been ascertained. Preliminary results highlight a general homogeneity between the two populations, nevertheless showing significant differences in some parameters (fruit form, pollen size). For this reason, and considering the geographic disjunction of the Sicilian plants, the two populations are proposed to be treated as two different subspecies: F. nodosa subsp. nodosa in the east and F. nodosa subsp. rigida (Ten.) Troia & Raimondo comb. & stat. nov. in the west. The names Ferula rigida Ten. and Ferula geniculata Guss. are also typified.

Sterility of Miscanthus × Giganteus Results from Hybrid Incompatibility

Miscanthus ×giganteus Greef et Deu. (Poaceae), a hybrid of Miscanthus sinensis and M. sacchariflorus native to Japan, is an ornamental and a highly lignocellulosic bioenergy crop, cultivated in the European Union as an alternative source of energy. This grass reproduces exclusively vegetatively, by rhizomes or via expensive in vitro micropropagation. The present study was aimed at finding the barriers that prevent sexual seed production, based on detailed embryological analyses of the whole generative cycle, including microsporogenesis, pollen viability, megasporogenesis, female gametophyte development, and embryo and endosperm formation. Sterility of M. ×giganteus results from abnormal development of both male and female gametophytes. Disturbed microsporogenesis (laggard chromosomes, univalents, micronuclei) was further highlighted by low pollen staining. The frequency of stainable pollen ranged from 13.9% to 55.3% depending on the pollen staining test, and no pollen germination was observed either in vitro or in planta. The wide range of pollen sizes (25.5–47.6 μm) clearly indicated unbalanced pollen grain cytology, which evidently affected pollen germination. Only 9.7% of the ovules developed normally. No zygotes nor embryos were found in any analyzed ovules. Sexual reproduction of M. ×giganteus is severely hampered by its allotriploid (2n=3x=57) nature. Hybrid sterility, a strong postzygotic barrier, prevents sexual reproduction and, therefore, seed formation in this taxon.

Hybridization amongst New Zealand Schoenus (Cyperaceae)?

Artificial pollinations were made between five New Zealand species of Schoenus (Cyperaceae) to determine whether they could form interspecific hybrids. No hybrids were produced and the site of the interspecific incompatibility was investigated using fluorescence microscopy. The site of pollen inhibition varied in the different cross combinations. No relationship was observed between pollen size and subsequent pollen germination and tube growth in the style. The results do not support the claim that S. concinnus and S. nitens form hybrids in nature.

Pollen studies of Jatropha (Jatropha curcas L.)

The present investigation was undertaken at the Farm of All India Coordinated Research Project on Under Utilized Crops, Regional Research Station, S.D. Agricultural University, Sardarkrushinagar during the year 2002 and 2003. Pollen grains were creamish yellow and sticky. They were Spheroidal or globular in distilled water and acetocarmine with three to four germspores. The mean pollen size in distilled water was 90.5 and 83.5 μm in lower and upper tier of stamen, respectively, while in acetocarmine 82 and 89 μm in upper and lower tier of stamens, respectively. The pollen viability was quite high (95.04 to 98.77%). Maximum pollen germination (35.49%) and pollen tube length (265.19 μ) was recorded in 15 % sucrose solution with 0.005% boric acid.

Anthropogenic climate change and allergen exposure: The role of plant biology

Accumulation of anthropogenic gases, particularly CO(2), is likely to have 2 fundamental effects on plant biology. The first is an indirect effect through Earth's increasing average surface temperatures, with subsequent effects on other aspects of climate, such as rainfall and extreme weather events. The second is a direct effect caused by CO(2)-induced stimulation of photosynthesis and plant growth. Both effects are likely to alter a number of fundamental aspects of plant biology and human health, including aerobiology and allergic diseases, respectively. This review highlights the current and projected effect of increasing CO(2) and climate change in the context of plants and allergen exposure, emphasizing direct effects on plant physiologic parameters (eg, pollen production) and indirect effects (eg, fungal sporulation) related to diverse biotic and abiotic interactions. Overall, the review assumes that future global mitigation efforts will be limited and suggests a number of key research areas that will assist in adapting to the ongoing challenges to public health associated with increased allergen exposure.

Diversity and evolution of pollen-ovule production in Cuscuta (dodders, Convolvulaceae) in relation to floral morphology

Cuscuta (dodder, Convolvulaceae) is a genus of about 200 species of obligate stem parasites with subcosmopolitan distribution. The diversity of pollen and ovule production was surveyed in 128 species and ten varieties. Taxa were assigned to Cruden’s mating system categories based on their pollen-ovule ratios. Variation and correlations among floral characters were analyzed using regression and ANOVA, while the mating system categories were subjected to a linear discriminant and canonical variates analysis to assess their cohesiveness. Our data strongly suggest that most Cuscuta species possess a wide range of mixed-mating systems. Whereas four ovules develop in each flower, pollen production varies over three orders of magnitude. Several Cuscuta taxa are highly outcrossing, but no species could be identified that are exclusively selfing. The transition from the one-style flowers of subg. Monogynella to the two-style flowers of subgenera Cuscuta and Grammica, and from simultaneous to sequential maturation of the two stigmas in the latter subgenus, has decreased the role of herkogamy as a facilitator of outcrossing. These evolutionary changes are associated with an increase of species richness in subgenus Cuscuta, and especially in subgenus Grammica. Morphological features were not individually found to have a strong correlation to the mating system, but in general, larger corollas and stigmas were associated with greater pollen-ovule ratios. Cuscuta presents some puzzling results when considered in light of the sex allocation theory, as only some infrageneric lineages demonstrate the predicted pollen size-number tradeoff, while Cuscuta gracillima complex (in subgenus Grammica) displays an unexpected negative relationship between pollen size and style length. The relationship between host range and mating system is discussed, prompting further research into the co-evolution of pollination systems and life history traits between parasites and their host species.

Pollen Viability and Longevity of Switchgrass (Panicum virgatum L.)

ABSTRACTPollen is essential for seed production and serves as the primary means of gene flow in outcrossing species like switchgrass (Panicum virgatum L.). There is a lack of information on basic pollen biology in switchgrass. This study investigated pollen viability, pollen longevity, and pollen size using different materials, including the tetraploid cultivar Alamo, the octoploid cultivar Cave‐in‐Rock, and transgenic Alamo plants. Pollen grains were collected from field‐grown Alamo and Cave‐in‐Rock plants, and greenhouse‐grown transgenics. Pollen size was in the range of 42.5 to 54.0 μm; no significant difference was observed in average pollen size between transgenic and control plants. Increasing temperature and ultraviolet‐B irradiation negatively affected pollen viability and longevity, while relative humidity had only limited impact. Weather conditions had a large impact on pollen longevity. Under sunny atmospheric conditions, pollen longevity of both cultivars decreased rapidly, with a half‐life of &lt;4.9 min and a complete loss of viability in 20 min. Under cloudy atmospheric conditions, the half‐life of pollen was more than fivefold longer than under sunny conditions, and it took approximately 150 min to lose viability completely. No difference in pollen viability and longevity was found between transgenic and nontransgenic control plants.

Asymmetrical gene flow in a hybrid zone of Hawaiian Schiedea (Caryophyllaceae) species with contrasting mating systems.

Asymmetrical gene flow, which has frequently been documented in naturally occurring hybrid zones, can result from various genetic and demographic factors. Understanding these factors is important for determining the ecological conditions that permitted hybridization and the evolutionary potential inherent in hybrids. Here, we characterized morphological, nuclear, and chloroplast variation in a putative hybrid zone between Schiedea menziesii and S. salicaria, endemic Hawaiian species with contrasting breeding systems. Schiedea menziesii is hermaphroditic with moderate selfing; S. salicaria is gynodioecious and wind-pollinated, with partially selfing hermaphrodites and largely outcrossed females. We tested three hypotheses: 1) putative hybrids were derived from natural crosses between S. menziesii and S. salicaria, 2) gene flow via pollen is unidirectional from S. salicaria to S. menziesii and 3) in the hybrid zone, traits associated with wind pollination would be favored as a result of pollen-swamping by S. salicaria. Schiedea menziesii and S. salicaria have distinct morphologies and chloroplast genomes but are less differentiated at the nuclear loci. Hybrids are most similar to S. menziesii at chloroplast loci, exhibit nuclear allele frequencies in common with both parental species, and resemble S. salicaria in pollen production and pollen size, traits important to wind pollination. Additionally, unlike S. menziesii, the hybrid zone contains many females, suggesting that the nuclear gene responsible for male sterility in S. salicaria has been transferred to hybrid plants. Continued selection of nuclear genes in the hybrid zone may result in a population that resembles S. salicaria, but retains chloroplast lineage(s) of S. menziesii.

Pollen grain size variation in Caryophylloideae: a mixed strategy for pollen deposition along styles with long stigmatic areas?

The literature supports a positive correlation between pollen size and style length and that pollen size may be affected by a trade-off with pollen number. A correlation between pollen size and pollinator type has been hypothesized by some authors but not confirmed. To assess these associations, pollen grain number, pollen grain diameter and style length were measured in 53 species of Caryophylloideae that had been classified beforehand as being diurnal, nocturnal or self-pollinated. Surprisingly, many species showed two, and a few even three, distinct pollen grain size classes per individual anther. Therefore, further analyses were based on the different pollen size classes and respective pollen grain numbers. Firstly, we found no indication of a trade-off between pollen size and number. Secondly, a positive linear correlation between style length and pollen grain diameter was found using conventional analysis methods and phylogenetically independent contrasts. Thirdly, when using conventional analysis methods a strong relationship between style length and pollination mode was found, with nocturnal species having significantly longer styles and larger pollen grains than diurnal and selfing species. These results were not supported, however, when calculating comparisons of phylogenetically independent pairs. We discuss our findings in relation to intra-specific pollen polymorphism and pollen degeneration, both of which have been associated with different pollen grain sizes in Caryophylloideae. Furthermore, we discuss the possibility that pollen polymorphism might be a mixed strategy to optimise the success of pollen grains deposited at different pollen–ovule distances on long styles.

Does pollen aerodynamics correlate with pollination vector? Pollen settling velocity as a test for wind versus insect pollination among cycads (Gymnospermae: Cycadaceae: Zamiaceae)

Studies of cycad pollination globally have revealed obligate pollination mutualisms with insects, typically hostspecific beetles. Some cycads in the genus Cycas have floating seeds allowing oceanic dispersal, and have colonized remote islands such as Fiji, Guam, and Madagascar. This seems to contradict the expected requirement that specific beetle pollinators be present in order for cycads to establish in remote localities. We therefore postulated that these ‘island’ cycads are wind pollinated. We tested for adaptations associated with wind pollination in a laboratory study of pollen aerodynamics. The pollen from a range of (non-cycad) wind-pollinated plants was not relatively ‘buoyant’: the settling velocities of wind-pollinated species exceeded those of insect-pollinated cycads and some animal-pollinated angiosperms. We attributed this result to settling velocity being, in part, correlated with pollen size, as cycad pollen was consistently smaller. A better predictor for pollination vector was the tendency of pollen to aggregate during a vertical fall. Pollen of wind-pollinated plants separated into single grains, whereas cycad pollen and pollen of animal-pollinated angiosperms adhered in large aggregations, the settling velocity of which increased rapidly with aggregate size. The pollen of cycads known to be insect pollinated was similar in morphology, settling speed, and clumping behaviour to pollen of ‘island’ cycads of unknown pollination biology. Based on these aerodynamics, we predict that cycad species on oceanic islands have insect pollinators. More broadly, we propose that pollen aggregation during a vertical fall provides a sound test for wind versus animal pollination when the pollination vector is unknown.

Quantification of UV-B flux through time using UV-B-absorbing compounds contained in fossil Pinus sporopollenin

UV-B radiation currently represents c. 1.5% of incoming solar radiation. However, significant changes are known to have occurred in the amount of incoming radiation both on recent and on geological timescales. Until now it has not been possible to reconstruct a detailed measure of UV-B radiation beyond c. 150 yr ago. Here, we studied the suitability of fossil Pinus spp. pollen to record variations in UV-B flux through time. In view of the large size of the grain and its long fossil history, we hypothesized that this grain could provide a good proxy for recording past variations in UV-B flux. Two key objectives were addressed: to determine whether there was, similar to other studied species, a clear relationship between UV-B-absorbing compounds in the sporopollenin of extant pollen and the magnitude of UV-B radiation to which it had been exposed; and to determine whether these compounds could be extracted from a small enough sample size of fossil pollen to make reconstruction of a continuous record through time a realistic prospect. Preliminary results indicate the excellent potential of this species for providing a quantitative record of UV-B through time. Using this technique, we present the first record of UV-B flux during the last 9500 yr from a site near Bergen, Norway.

Reproductive mechanisms and pollen characterization in some accessions of an underutilized legume: (Sphenostylis stenocarpa Hochst Ex. A. Rich) harms

Studies on flowering, pollen, pod and seed characters, germination rate and seed set percentage were carried out on twenty five accessions of African Yam Bean (AYB). Self compatibility tests confirmed all the accessions as obligate selfers, while the reciprocal crosses were not successful. Germination rate, percentage seed set and pollen fertility were observed to be high in the accessions. Correlation analysis among the reproductive traits revealed that pod length, pod width, number of locules per pod and number of seeds per pod contributed significantly to the percentage seed set in all the accessions. Pollen fertility and seed viability were also observed to be high in all the accessions except in TSs23 where low pollen fertility, low seed viability and low percentage seed set were recorded. All the accessions produced tricolporate pollen grains. Pollen size ranged from 66.15 μm in TSs40 to 82.75 μm in TSs119, pollen fertility ranged from 53.39% in TSs23 to 95.30% in TSs119, seed set ranged from 83.32% in TSs90, 89.21% in TSs23 and 95.72% in TSs3. The germination rate ranged from 59.68% in TSs23 to 99.03% in TSs22. The percentage moisture content ranged between 4.38% in TSs22 and 11.43% in TSs119. Key words: Pollen characters, seed set, germination rate, self compatibility.

Pollen morphology of the family Cistaceae in Egypt and its systematic significance

Abstract The pollen morphology of 11 species (including two subspecies and two varieties) belonging to two genera (Helianthemum and Fumana) of the family Cistaceae in Egypt was studied using light and scanning electron microscopy. Pollen grains of the studied taxa were found to be radially symmetrical and tricolporate. Pollen size, shape, apertures, and exine ornamentation characteristics were valuable parameters among the studied taxa. The largest pollen size was recorded in H. salicifolium and the smallest one observed in H. kahiricum subsp. schweinfurthii. Pollen shape in the Egyptian taxa varied from (sub-)prolate to prolate spheroidal, but F. arabica is different in having sub-oblate grains. The pollen data confirm that H. lippii and H. sessiliflorum are very closely related species. Pollen sculpture was useful in distinguishing between H. vesicarium var. vesicarium and H. vesicarium var. ciliatum. Three main pollen types of exine ornamentation were recognized: retipilate; reticulate to verrucate; and striate. Based on palynological data, a key for the studied taxa is suggested.

The size and germinability of Scots pine pollen in different temperatures in vitro

The effect of genotype, the origin of genotype, and germination temperature on Scots pine pollen grain size, hydration rate, germinability, and tube growth was studied in vitro. The mean sizes of dry and germinated pollen grains varied among pollen genotypes in different ways, thus the hydration rate varied among genotypes. Pollen from Scots pine that originates in northern Finland hydrated more than pollen from a population in southern Finland. Germination temperature had no effect on the hydration rate. Germinability and tube growth rate of northern genotypes were higher at 20 °C than at 15 °C. Differences among southern genotypes were not significant. At 15 °C, the germinability and pollen tube growth rate of northern genotypes were lower than southern genotypes. At 20 °C, the differences were not significant. It appears that germination and growth of pollen from northern populations are enhanced at higher temperatures whereas pollen from southern populations is unaffected.