Pink Pigment Research Articles

Sir Joshua Reynolds’ Lady Caroline Howard

Sir Joshua Reynolds’ <i>Lady Caroline Howard</i>

HF 2EPR investigation of a Cr-bearing gahnite pigment

A pink gahnite pigment, containing the chromophore ion Cr 3+, has been synthesised under industrial conditions and characterised by X-ray powder diffraction (XRPD), electronic spectroscopy, electron paramagnetic resonance spectroscopy (EPR) and HF 2EPR. The paramagnetic resonance techniques, in particular, have been applied to determine the incorporation of the chromophore in the spinel structure and to study its distribution. The pigment was found to be formed by aluminium borate and, mainly, gahnite (ZnAl 2O 4). The Cr 3+ ion was revealed to be incorporated only in the octahedral site of this latter phase. The chromophore is subjected to a strong crystal field ( Δ 0 = 18800 cm −1), which determines its colouring properties. A large zero field splitting interaction was observed in the X-band EPR spectra and interpreted by comparison with the high-frequency W-band spectra. The axial symmetry of the crystal field surrounding Cr 3+ was ascertained by both the techniques. The observed spectroscopic features are due to the single ion properties of Cr 3+ in a strong field, thus ruling out any possible clustering of the chromophore within the structure.

Allium discoloration: precursors involved in onion pinking and garlic greening.

Precursors involved in the formation of pink and green-blue pigments generated during onion and garlic processing, respectively, have been studied. It has been confirmed that the formations of both pigments are of very similar natures, with (E)-S-(1-propenyl)cysteine sulfoxide (isoalliin) serving as the primary precursor. Upon disruption of the tissue, isoalliin and other S-alk(en)ylcysteine sulfoxides are enzymatically cleaved, yielding 1-propenyl-containing thiosulfinates [CH3CH=CHS(O)SR; R = methyl, allyl, propyl, 1-propenyl] among others. The latter compounds have been shown to subsequently react with amino acids to produce the pigments. Whereas the propyl, 1-propenyl, and methyl derivatives form pink, pink-red, and magenta compounds, those containing the allyl group give rise to blue products after reacting with glycine at pH 5.0. The role of other thiosulfinates [RS(O)SR'] (R, R' = methyl, allyl, propyl) and (Z)-thiopropanal S-oxide (the onion lachrymatory principle) in the formation of the pigments is also discussed.

The influence of iron content on the promotion of the zircon structure and the optical properties of pink coral pigments

A sol–gel reaction starting from Si and Zr alkoxides, in water-ethanol mixtures, was employed to obtain iron doped zirconium silicate powders (zircon). The starting amount of the ferric salt in the sol–gel reacting mixture was varied in order to obtain Fe 2O 3/Zr molar ratios in the range 0.7–10%. The products of the sol–gel reaction were calcined in the range 800–1300 °C. X-ray diffractograms, EDX analyses and diffuse reflectance spectra were obtained and analysed for all the calcined powders; the colour of the pigments was characterised on the grounds of the CIE (Commission Internationale de l’Eclairage) standard procedure (CIE L ∗ a ∗ b ∗ measurements). Results from the structural and spectral characterisations are examined and cross-compared to produce a consistent picture of the role played by iron on the promotion of the zircon lattice and on the optical properties of the reaction products.

Plant colonization by pink-pigmented facultative methylotrophic bacteria (PPFMs)

Bacteria belonging to the genus Methylobacterium are characterized by being able to rely on methanol as a sole carbon and energy source and by presenting a more or less intense pink reddish pigmentation. These bacteria, also referred to as pink-pigmented methylotrophic bacteria (PPFMs), are common inhabitants of the phyllosphere and are found in many other environmental samples. Since they grow slowly they are often overlooked and their impact on phyllosphere microbial communities and on the plants harboring them is not well studied nor has their ecology been elucidated. In a survey of PPFM colonization in three different agricultural sites, PPFM populations were identified on both red clover and winter wheat, but red clover was more consistently colonized. Isolations from collected leaves showed PPFM populations to decrease from spring towards summer, but they increased again towards the end of the cropping season. Isolates from red clover readily colonized winter wheat leaves and vice versa in greenhouse experiments, but population sizes were dependent on the application procedure. Tested isolates had also good potential to colonize the rhizosphere, especially after seed inoculations. Confocal scanning laser microscopy showed gfp-tagged isolates to colonize the surface of clover leaves by forming large aggregates.

Aerosol-derived Mn-doped Al 2O 3 pink pigments prepared in the absence of fluxes

A method for the preparation of Mn-doped alumina pink pigments in the absence of fluxes through the pyrolysis of liquid aerosols generated from aluminium(III) nitrate and manganese(II) nitrate aqueous solutions is reported and compared with the traditional ceramic procedure. It was found that the pyrolysis method requires a lower temperature (900 °C) for the development of the corundum phase and the pink colour than that required by the traditional procedure (1300 °C) yielding a more intense colour, as a consequence of the incorporation of a higher amount of Mn in solid solution to the alumina lattice. In addition, the use of the pyrolysis procedure allows the preparation of pigments grains with uniform shape and controlled and reproducible size distribution, not requiring grinding.

Raman investigation of a pink pigment on a shard from the São João Baptista shipwreck

Raman investigation of a pink pigment on a shard from the São João Baptista shipwreck

S-Substituted cysteine derivatives in production of flavour and colour

A brief review of our continuous investigations on S-substituted cysteine derivatives is presented herein. It comprises a summary on nonenzymatic (thermal) decomposition of these amino acids as well as on their role in the formation of blue and pink pigments during processing of garlic and onion, respectively. The emphasis is put on four most common derivatives, namely S-methyl-, S-allyl-, S-1-propenyl- and S-propylcysteine sulfoxides (methiin, alliin, isoalliin and propiin, respectively). Our results demonstrate that these sulfur-containing amino acids are the key primary precursors in the formation of both flavour and colour of alliaceous plants.

Allium discoloration: the nature of onion pinking and garlic greening

Precursors involved in the formation of pink and green-blue pigments during onion and garlic processing, respectively, have been studied. It has been confirmed that the formation of both pigments is of a very similar nature, with (E)-S-(1-propenyl)cysteine sulfoxide (isoalliin) serving as the primary precursor. Upon disruption of the tissue, isoalliin and other S-alk(en)ylcysteine sulfoxides are enzymatically cleaved, yielding prop-1-enylcontaining thiosulfinates [CH&lt;sub&gt;3&lt;/sub&gt;CH = CHS(O)SR; R = methyl, allyl, propyl, 1-propenyl], among others. The latter compounds subsequently react with amino acids to produce the pigments. Whereas the onion and leek-related propyl, prop-1-enyl and methyl derivatives can form pink, pink-red and magenta compounds, those containing the allyl group yield dark blue products after reacting with glycine at pH 5.0.

First Report of Pink Seed of Lentil and Chickpea Caused by Erwinia rhapontici in Canada.

A new disease of lentil (Lens culinaris Medik.) and chickpea (Cicer arietinum L.) caused by Erwinia rhapontici (Millard) Burkh. was found in seed samples from commercial fields in Saskatchewan, Canada in 2002. Infected seeds had a pink or pinkish-brown discoloration of the seed coat. Isolation from surface-sterilized pink seeds resulted in bacterial cultures that produced a water-soluble pink pigment on potato dextrose agar (PDA). Four isolates from different lentil crops, LRC 8265, LRC 8310, LRC 8309, and LRC 8313 and one isolate from a chickpea crop, LRC 8266, were tested as previously described (2). Results of the tests were identical to those for pink bean isolates of E. rhapontici (2) with the following minor exceptions: all were negative for Voges-Proskauer; LRC 8266 was positive for tagatose; LRC 8266, LRC 8309, and LRC 8313 were negative for lactose; and LRC 8266 and LRC 8309 were positive for 5-keto gluconate. For pathogenicity tests, each isolate was inoculated into 30 pods from 6 lentil plants (cv. Laird), 30 pods from 6 desi chickpea plants (cv. Myles), and 30 pods from 6 kabuli chickpea plants (cv. Sanford) by the method described for pink seed of pea (1) and bean (2). Each pod was inoculated with 0.1 ml (0.2 ml for kabuli chickpeas) of bacterial suspension, approximately 108 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations of the pathogen were performed as described above. In duplicate experiments, all the isolates caused pink lesions on pods and seeds of lentil, desi chickpea, and kabuli chickpea. The frequency of infected seeds among the five isolates (four lentil and one chickpea) ranged from 50 to 100% on lentil, 73 to 100% on desi chickpea, and 43 to 100% on kabuli chickpea. E. rhapontici was reisolated from seeds with lesions but not from asymptomatic seeds. The study demonstrates that in addition to pea (1) and common bean (2), E. rhapontici is also the causal agent of pink seed of lentil and chickpea. The observation that lentil isolates can infect chickpea and vice versa suggests that host specificity may be lacking in E. rhapontici. To our knowledge, this is the first record of E. rhapontici on lentil and chickpea.

Pink pigment optimization by resonance Raman spectroscopy

AbstractThe chromium‐doped tin‐based sphene (malayite) pink pigment was synthesized in different ways in the form of powder and single crystals. The usefulness of resonance Raman spectroscopy as a fast characterization technique for such ceramic pigments was evaluated with emphasis on the colour shift induced by second phases. Copyright © 2003 John Wiley &amp; Sons, Ltd.

Demonstration of a sensory rhodopsin in eubacteria.

We report the first sensory rhodopsin observed in the eubacterial domain, a green light-activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all-trans retinal to form a pink pigment (lambda max 543 nm) with a photochemical reaction cycle of 110 ms half-life (pH 6.8, 18 degrees C). Co-expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane-embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS-PAGE and autofluorography of 3H-labelled all-trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix-helix interactions with membrane-embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.

Encapsulation of Hematite in Zircon by Microemulsion and Sol-Gel Methods

Microemulsion method has been applied and compared with sol-gel, coprecipitation and ceramic route with the purpose of encapsulating hematite into zircon crystals used as ceramic pigment. In the commercial synthesis of pink coral pigment, NaF flux agent is usually added in order to improve the reactivity of the system; therefore, the addition of flux agents (NaF or NaF · 2NaCl) has been investigated. Likewise, the effect of precipitating agent (NH3 or NaOH) has been studied. Sol-gel becomes the more reactive method and produces the best red colours without fluorides addition, but does not give red colour in the presence of halides fired at 1000°C. Ammonia microemulsioned and coprecipitated powders give similar orange-brown colours without fluorides, and red colours with fluorides addition. The addition of NaF produces higher inclusion effectiveness than NaF · 2NaCl. The stabilization of sodium silicozirconate crystalline phases in NaOH coprecipitated or emulsioned samples avoids the zircon crystallization and then the hematite encapsulation.

O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction.

The nature and specificity of the Wiesner test (phloroglucinol-HCl reagent) for the aromatic aldehyde fraction contained in lignins is studied. Phloroglucinol reacted in ethanol-hydrochloric acid with coniferyl aldehyde, sinapyl aldehyde, vanillin, and syringaldehyde to yield either pink pigments (in the case of hydroxycinnamyl aldehydes) or red-brown pigments (in the case of hydroxybenzaldehydes). However, coniferyl alcohol, sinapyl alcohol, and highly condensed dehydrogenation polymers derived from these cinnamyl alcohols and aldehydes did not react with phloroglucinol in ethanol-hydrochloric acid. The differences in the reactivity of phloroglucinol with hydroxycinnamyl aldehydes and their dehydrogenation polymers may be explained by the fact that, in the latter, the unsubstituted (alpha,beta-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the beta carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuumvar. annuum, Populus albaL., and Pinus halepensisL. demonstrated the presence of 4- O-linked hydroxycinnamyl aldehyde end groups and 4- O-linked 4-hydroxy-3-methoxy-benzaldehyde (vanillin) end groups in lignins. However, given the relatively low abundance of 4- O-linked vanillin in lignifying cell walls and the low extinction coefficient of its red-brown phloroglucinol adduct, it is unlikely that vanillin contributes to a great extent to the phloroglucinol-positive stain reaction. These results suggest that the phloroglucinol-HCl pink stain of lignifying xylem cell walls actually reveals the 4- O-linked hydroxycinnamyl aldehyde structures contained in lignins. Histochemical studies showed that these aldehyde structures are assembled, as in the case of coniferyl aldehyde, during the early stages of xylem cell wall lignification.

Synthesis of chromium-containing pigments from chromium recovered from leather waste

Cobalt-chromite green and chrome-tin pink pigments have been prepared from chromium extracted from leather shavings produced as a waste product of the leather-tanning industry. The alkaline agent (NaOH, CaO, MgO, NH 4OH) used in the extraction process influences the nature of the final product. The effect of the NH 4OH:CaO ratio on the final product in the case of cobalt-chromite green was examined. The pigments obtained were characterized by FT-IR and X-ray diffraction (XRD). Colour measurements were compared with those recorded from materials prepared from pure Cr 2O 3. The leaching of Cr(VI) from the materials was examined by UV spectroscopy.

First Report of Pink Seed of Common Bean Caused by Erwinia rhapontici.

In 2001, a new disease of common bean (Phaseolus vulgaris L.) caused by Erwinia rhapontici (Millard) Burkh. was detected in seed samples from southern Alberta, Canada. Infected seeds had pink or pinkish-brown lesions on the seed coat. The disease was found in great northern (cv. US1140), pink (cv. Viva), and pinto (cv. Othello) beans at low (<0.1%) frequencies. Isolation from surface-sterilized pink seeds resulted in bacterial cultures, which produced a water-soluble pink pigment on potato dextrose agar (PDA). Seven isolates were tested for physiological characteristics using conventional tests (1) and API 50CHE test strips (bioMérieux Canada, St. Laurent, Quebec), and tested for cellular fatty acids using the MIDI system (Newark, DE). All isolates were gram-negative, motile, facultative anaerobic rods with mucoid colonies and produced a pink pigment on PDA. They were positive for citrate utilization, catalase, methyl red, and Voges-Proskauer, and negative for arginine dihydrolase, lysine and ornithine decarboxylases, urease, gelatin liquification, indole production, oxidase, and gas production. Fatty acid profiles matched with E. rhapontici (approximately 30% each 16:0 and 16:1 ω7c/15:0 iso 2OH; 12% 18:1 ω7c: 8% each 17:0 cyclo and 14:0 3OH/16:1 iso; 4 to 5% each 12:0 and 14:0). Isolates were positive for acid production from: N-acetyl glucosamine, l-arabinose, amygdalin, arbutin, cellobiose, esculin (hydrolysis), d-fructose, d-fucose, d-galactose, β-gentiobiose, d-glucose, glycerol, i-myo-inositol, lactose, maltose, d-mannitol, d-mannose, melibiose, d-raffinose, l-rhamnose, ribose, salicin, d-sorbitol, sucrose, trehalose, and d-xylose. These results match published results for E. rhapontici (4). For pathogenicity tests, each isolate was inoculated in 30 pods from six bean plants (cv. US1140) as described for pink seed of peas (2). Each pod was inoculated with 0.1 ml of bacterial suspension, approximately 109 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations were done as described above. In duplicate experiments, all isolates caused lesions on pods extending up to 5 cm from the inoculation point with corresponding discoloration of seeds. The frequency of infected seeds varied among isolates, ranging from 20 to 50%. E. rhapontici was reisolated from seeds with lesions, but not asymptomatic seeds. The study concludes that pink seed of common bean is due to E. rhapontici, a pathogen previously reported on peas in Alberta, Canada (2), and Montana (3).

Plants in the pink: cytokinin production by methylobacterium.

Bacteria of the genus Methylobacterium are well-studied examples of facultative methylotrophs. These bacteria are classified as α-proteobacteria and are capable of growth on methanol and methylamine as well as on a variety of C2, C3, and C4 compounds (8). Methylobacterium strains are commonly found in soils, as well as on the surfaces of leaves of a wide variety of plants (1). Because of their distinctive pink pigmentation, they are sometimes referred to as PPFMs (pink-pigmented facultative methylotrophs). Evidence exists that these bacteria utilize methanol emitted by the stomata of plants (9), but the details of their relationship to plants has been unclear. Are Methylobacterium strains commensal bacteria, or do they communicate with plants in a more intimate relationship? A number of reports suggest that Methylobacterium strains are more than passive passengers on plant leaves. For instance, they have been shown in some cases to stimulate seed germination and plant development, possibly by production of phytohormones (2, 3, 4), and it has been reported that one strain produces the cytokinin zeatin (5) while others have been reported to produce indole acetic acid (6). In addition, Methylobacterium strains have been suggested to contribute to the flavor of strawberries (12) and have been localized as endosymbionts within cells of the buds of Scotch pine (Pinus sylvestris) (10). One nonpigmented Methylobacterium strain has been shown to form a root-nodulating nitrogen-fixing symbiosis with a legume (11). A partial genome sequence of Methylobacterium extorquens AM1 is available (http://pedant.mips.biochem.mpg.de/), which reveals a number of open reading frames with significant identity to genes involved in plant association in rhizobia and Agrobacterium (M. Lidstrom, unpublished results). Therefore, at this point numerous indications exist that these PPFMs interact with plants, but the biochemical and genetic details of those interactions have remained elusive. In this issue, Koenig et al. (7) present a set of studies that represents an important step in understanding the molecular basis of Methylobacterium-plant interactions. They show that four different Methylobacterium strains representing leaf isolates and a type M. extorquens strain all produce the cytokinin trans-zeatin at low levels in pure culture and excrete it into the culture medium. Biochemical and genetic evidence is presented suggesting that the trans-zeatin is not synthesized de novo but instead is derived from tRNA. This result is surprising, since so far the tRNA-derived zeatin from bacteria and plants has been restricted to the cis isomer. Is the low-level production of trans-zeatin observed in these strains sufficient to have an effect on plants? Surprisingly, the Methylobacterium-specific stimulation of seed germination that has been described in the past (3, 4) was unaffected in a mutant that is incapable of producing trans-zeatin. Thus, the answer to this question is still outstanding, as is the source of the stimulation effect itself. However, the results presented by Koenig et al. provide mechanistic evidence for cytokinin production by Methylobacterium strains, leading the way for future studies of the Methylobacterium-plant relationship. In addition, this evidence that a commensal bacterium produces a phytohormone via a previously unknown route provides new insights into the role of commensal plant bacteria in general.

Liquid chromatography-mass spectrometry (LC-MS) investigation of the thiobarbituric acid reactive substances (TBARS) reaction.

The thiobarbituric acid reactive substances (TBARS) assay is a commonly used method for the detection of lipid peroxidation. Malondialdehyde is formed as a result of lipid peroxidation and reacts with thiobarbituric acid to form a pink pigment that has an absorption maximum at 532 nm. Other compounds also react with thiobarbituric acid to form colored species that can interfere with this assay, but little is known about these interfering species. This is the first investigation using LC-MS and MS-MS to study the structures of the pink adduct as well as a common unstable yellow interference compound, which absorbs at 455 nm. Also, the presence of barbituric acid impurities in the thiobarbituric acid reagent was found to produce 1:1:1 thiobarbituric acid/malondialdehyde/barbituric acid and 2:1 barbituric acid/malondialdehyde adducts that absorbed at 513 and 490 nm, respectively, indicating that thiobarbituric acid should be purified before use.

First Report of Pink Seed of Pea Caused by Erwinia rhapontici in the United States.

In March 2001, the USDA Federal Grain Inspection Service sent for analysis to USDA-ARS, Pullman, WA, 12 discolored seeds of field pea (Pisum sativum L.) from northeastern Montana. Symptoms consisted of pale pinkish brown-to-bright pink discoloration throughout the seed coat. Unlike the pink coloration resulting from application of pink pesticidal seed treatments, the coloration was permanent and could not be removed by extended washing. Ten discolored seeds were disinfested in 0.5% NaOCl for 1 to 2 min and rinsed in sterile distilled water. Five seeds were placed on malt extract agar amended with streptomycin sulfate and tetracycline hydrochloride at 50 mg per liter each, and five seeds were placed on nutrient broth yeast extract agar (NBY) (3) and incubated under ambient lab conditions. No organisms were isolated from peas on the antibiotic agar, but pale pink bacterial colonies were recovered from each of the five peas on NBY. The pink colonies were streaked for selection of single colonies, stored at 4°C, and revived for growth in shaken (120 rpm) NBY broth to optical density (OD) = 0.1 at 640 nm (≈108 CFU/ml). Three pea plants and one control plant were grown in the greenhouse until pods were sufficiently developed to be syringe-inoculated at the suture with 100 μl of bacterial culture. The experiment was repeated using a second bacterial isolate. In each experiment, pods on inoculated plants, but not the control plant, exhibited pale pinkish areas and later produced seeds discolored in shades of pink matching those from the original sample. Some symptomatic seeds were shriveled or aborted as well as discolored. Discolored seeds from the two experiments were plated to NBY and pale pinkish colonies were recovered as before, but no such colonies were recovered from the controls. Two isolates from the original sample and two recovered from the experimental inoculations were tested for anaerobic and aerobic growth using API 50 CHE and API 20 NE tests (bio Mérieux Vitek, Inc., Hazlewood, MO). Known strains of Erwinia rhapontici and E. rubrifaciens from the collection of Dennis Gross (Texas A&M University) were identically analyzed for comparison. All four isolates from pea were gram-negative, facultatively anaerobic, motile rod-shaped bacteria that produced a diffusible pink pigment on NBY. Tests indicated that these strains were E. rhapontici, because the results agreed with previously published data (1,2). Results with the known strain of E. rhapontici were congruent with those from the pea strains. The strain of E. rubrifaciens also produced a pink pigment on NBY, but unlike the other strains, it grew well at 37°C and was negative for acetoin production, nitrate reduction, esculin hydrolysis, and maltose, rhamnose, inositol, and melibiose fermentation and did not assimilate citrate aerobically. Pink seed of pea has been previously reported from Alberta, Canada (2).

Notes and New Species of Moraea (Iridaceae: Iridoideae) from the Southern African Winter-Rainfall Zone

Moraea cantharophila and M. lilacina are new species of the African genus Moraea (ca. 195 species), of subfamily Iridoideae of the Iridaceae. Both species belong to Moraea subg. Vieusseuxia, which comprises 35 species restricted to southern Africa and is centered in the southern African winter-rainfall zone. Moraea cantharophila, from southwestern Western Cape Province, has unusual flowers for the subgenus with entire relatively large inner tepals with a broad dark median streak on the claw. It is distinguished from its closest relative, the fly-pollinated M. lurida, by the shallower floral cup and unscented nectarless flowers. Moraea lilacina, from the northern Langeberg foothills in the Little Karoo of southeastern Western Cape Province, can be distinguished from the related M. unguiculata by its relatively large flower, pink pigmentation, trilobed inner tepals with a long, erect central cusp, and often densely hairy filament column. Nomenclatural adjustments include the reduction of M. neopavonia in M. tulbaghensis and the provision of a new name for the homonym M. flexuosa Goldblatt. Newly discovered populations of M. saxicola and M. rivulicola increase their known geographical ranges and require some adjustment to their delimitation.