Floral pigments and their perception by avian pollinators in three Chilean Puya species

doi:10.21203/rs.3.rs-3579433/v1

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Floral pigments and their perception by avian pollinators in three Chilean Puya species

https://doi.org/10.21203/rs.3.rs-3579433/v1

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published 04 Mar, 2024

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The Chilean Puya species, Puya coerulea var. violacea and P. chiliensis bear blue and pale-yellow flowers, respectively, while P. alpestris considered to be their hybrid-derived species has unique turquoise flowers. In this study, the chemical basis underlying the different coloration of the three Puya species was explored. We first isolated and identified three anthocyanins: delphinidin 3,3′,5′-tri-O-glucoside, delphinidin 3,3′-di-O-glucoside and delphinidin 3-O-glucoside; seven flavonols: quercetin 3-O-rutinoside 3′-O-glucoside, quercetin 3,3′-di-O-glucoside, quercetin 3-O-rutinoside, isorhamnetin 3-O-rutinoside, myricetin 3,3′,5′-tri-O-glucoside, myricetin 3,3′-di-O-glucoside and laricitrin 3,5′-di-O-glucoside; and six flavones: luteolin 4′-O-glucoside, apigenin 4′-O-glucoside, tricetin 4′-O-glucoside, tricetin 3′,5′-di-O-glucoside, tricetin 3′-O-glucoside and selagin 5′-O-glucoside from their petals. We also compared compositions of floral flavonoid and their aglycone among these species, which suggested that the turquoise species P. alpestris has an essentially intermediate composition between the blue and pale-yellow species. The vacuolar pH was relatively higher in the turquoise (pH 6.2) and pale-yellow (pH 6.2) flower species, while that of blue flower species was usual (pH 5.2). The flower color was reconstructed in vitro using isolated anthocyanin, flavonol and flavone at neutral and acidic pH, and its color was analyzed by reflectance spectra and the visual modeling of their avian pollinators. The modeling demonstrated that the higher pH of the turquoise and pale-yellow species enhances the chromatic contrast and spectral purity. The precise regulation of flower color by flavonoid composition and vacuolar pH may be adapted to the visual perception of their avian pollinator vision.

anthocyanins

Bromeliaceae

color turning

intermolecular co-pigmentation

vacuole pH

Turquoise color development of flowers is rare in nature, and such the unique trait needs to be understood and discussed how it was acquired (Iwashina et al., 2015; Okitsu et al., 2018). Puya alpestris (Poepp.) Gay (Bromeliaceae), endemic to Chile (Schulte et al., 2010; Zizka et al., 2013), bears turquoise flowers. Anthocyanin, delphinidin 3,3′,5′-tri-O-glucoside, flavones, apigenin 4′-O-glucoside and luteolin 4′-O-glucoside, and flavonols, myricetin 3,3′,5′-tri-O-glucoside and myricetin 3-O-rutinoside 3′,5′-di-O-glucoside have been reported as its flower pigments (Mizuno et al., 2021). Moreover, it has been revealed that the turquoise color expression is developed by co-expression of blue color due to intermolecular copigmentation between their anthocyanin and flavones, and pale-yellow color expression from the flavonols under relatively higher pH (Scogin and Freeman, 1984; Scogin, 1985; Mizuno et al., 2021).

Molecular phylogenetic studies of Puya have revealed that P. alpestris has a sister relationship with Chilean Puya species and suggested that the turquoise flowered species is derived from a hybrid between blue flowered P. coerulea Lindl. and/or P. venusta, (Baker) Phil., and pale-yellow flowered P. chilensis Molina and/or P. gilmartiniae G.S.Varad. & A.R.Flores (Jabaily and Sytsma, 2010). In addition, ecological studies of Chilean Puya species have showed the relationship between their avian pollinators and morphology and nectar characteristics of these flowers (Hornung et al., 2013). According to Hornung et al. (2013), blue flowered species P. coerulea and P. venusta are mainly visited by specialist hummingbirds, having highly concentrated nectar in relatively low volume and no sterile apex in the inflorescence. On the other hand, P. alpestris and P. chilensis have lower nectar concentration in large nectar volume and sterile apexes, which are exclusively visited by generalist passerines (Hornung et al., 2013). These pollinators of the three Puya species include both visual sensory types, ultraviolet-sensitive (UVS) and violet-sensitive (VS) type (Ödeen and Håstad, 2010).

In this study, to understand the chemical basis in natural history of acquisition of the unique flower color trait, the flower pigments, anthocyanins, flavones and flavonol were isolated and identified from the flower of bluish P. coerulea var. violacea, turquoise P. alpestris, and pale-yellow P. chilensis. We then compared the flavonoid composition among these Chilean Puya species to examine the chemical factor lying on their different floral coloration. Color parameters of these Puya flowers were also evaluated based on the visual models of UVS- and VS-avian pollinators, using in vitro reconstructed flower colors. Moreover, we discussed the possibility of generation of the turquoise flowered species by interspecific hybridization between blue flowered and pale yellow flowered Puya species.

Identification of the flavonoids.

Three anthocyanins (1–3), four flavonols (4–7) and three flavones (8–10) were isolated from the flowers of P. coerulea var. violacea. On the other hand, three flavonols (11, 12 and 14) and three flavones (13, 15 and 16) were isolated from the flowers of P. chilensis. Their chemical structures are shown in Figs. 1A and B, and Table 1.

The liquid chromatography-mass spectrometry (LC/MS) measurement of three anthocyanins exhibited molecular ion peaks at m/z 789 [M]⁺ (1), 627 [M]⁺ (2) and 465 [M]⁺ (3). Fragment ion peaks corresponding to delphinidin were observed at m/z 303 [M]⁺ for all of 1–3. The UV–vis spectra of these anthocyanins showed the relatively high E₄₄₀/E_max values, 21–32%, meaning that the 5-hydroxyl group is free. In anthocyanin 3, bathochromic shift of the λ_max was observed by addition of AlCl₃, showing the presence of ortho-dihydroxyl group in the B-ring. Finally, they were identified as delphinidin 3,3′,5′-tri-O-glucoside (1, preternatin C5), delphinidin 3′,5′-di-O-glucoside (2) and delphinidin 3-O-glucoside (3, myrtillin) by HPLC and TLC comparisons with the authentic samples. Compound 1 has been reported from the flowers of P. alpestris as the main pigment (Mizuno et al., 2021).

Compound 4 showed the peaks of deprotonated molecule at m/z 771 [M − H]⁻ and fragment ions at m/z 625 [M − H−146]⁻ and 303 [M − H−162×2 − 146]⁻ on LC/MS. UV spectral properties indicated that the aglycone of 4 has free 5-, 7- and 4′-hydroxyl groups. In nuclear magnetic resonance (NMR) spectrum, five aromatic proton signals at δ_H 8.01 (d, H-2′), 7.92 (dd, H-6′), 6.94 (d, H-5′), 6.45 (brs, H-8) and 6.19 (brs, H-6) appeared (Table 2). The attachment of glucose to 3- and 3′-positions of aglycone was determined by HMBC correlation between glucosyl anomeric proton signals at δ_H 5.04 and 4.97, and C-3 and C-3′ signals at δ_C 134.2 and 146.5, respectively (Fig. S1). The β-pyranose form of the glucose was assigned by ¹H NMR and NOESY correlation (Fig. S2) (Markham and Geiger, 1994). Thus, 4 was identified as quercetin 3-O-rhamnopyranosyl-(1→6)-glucopyranoside-3′-O-glucopyranoside. The compound has been reported from the seeds of Fagopyrum tataricum (L.) Gaertn. (Polygonaceae) (Wu et al., 2007).

The peaks of deprotonated 5, 6 and 7 occurred at m/z 625, 609 and 623 [M − H]⁻, respectively. From the UV spectral properties, these compounds were compared by HPLC and TLC with authentic samples, and identified as quercetin 3,3′-di-O-glucoside (5), quercetin 3-O-rutinoside (6) and isorhamnetin 3-O-rutinoside (7).

The LC/MS of 8, 9 and 10 showed the peaks at m/z 447 and 433 [M + H]⁺ and 463 [M − H]⁻, respectively. Compounds 8 and 9 were identified as luteolin 4′-O-glucoside (8) and apigenin 4′-O-glucoside (9) by UV spectral properties and HPLC and TLC comparisons with authentic samples. Compound 8 has been isolated and identified as copigment substance for blue expression from the flowers of P. alpestris (Mizuno et al., 2021). Compound 10 was characterized as tricetin 4′-O-glucoside by UV and NMR spectra analysis after acid hydrolysis.

LC/MS of 11 detected the peaks of deprotonated molecule at m/z 803 [M − H]⁻ and three fragment ions at m/z 643 [M + H − 162]⁺, 481 [M + H − 162×2]⁺ and 319 [M + H − 162×3]⁺, meaning that 11 is myricetin attached to 3 mol hexoses. UV spectral properties showed that 11 is myricetin glycosylated at the 3-, 3′- and 5′-positions. Finally, it was determined as myricetin 3,3′,5′-tri-O-glucoside by HPLC and TLC comparisons with an authentic sample, which has been reported as pale-yellow pigment from the flowers of P. alpestris (Mizuno et al., 2021).

Compound 12 showed the peaks of deprotonated molecule at m/z 641 [M − H]⁻ and fragment ions at m/z 481 [M + H − 162]⁺ and 319 [M + H − 162×2]⁺ on LC/MS, showing that 12 is myricetin attached to 2 mol hexoses. UV spectral properties showed that 3- and 3′-positions of myricetin are glycosylated. In ¹H NMR spectrum, four aromatic proton signals, δ_H 7.63 (d, H-2′), 7.50 (d, H-6′), 6.41 (d, H-8) and 6.18 (d, H-6) appeared (Table 2). The attachment of the sugars to 3- and 3′-position of myricetin was determined by HMBC correlation between anomeric proton signals at δ_H 5.20 (d) and δ_H 4.87 (d) and C-3 and C-3′ signals at δ_C 135.7 and δ_C 146.9, respectively (Fig. S3). The β-pyranose forms of the glucose were assigned by ¹H NMR and NOESY correlation (Fig. S4). Thus, 12 was identified as myricetin 3,3′-di-O-glucopyranoside. The compound has been found in the leaves of Rhizophora mangle (Rhizophoraceae) (Kandil et al., 2004).

LC/MS of 13 showed the peaks at m/z 627 [M + H]⁺ and 625 [M − H]⁻ and the fragment ion peak at m/z 311 [M + H − 162×2]⁺, suggesting that 13 is tricetin attached to 2 mol hexoses. UV spectral properties showed that the presence of free 7- and 4′-hydroxyl groups. In ¹H NMR spectrum, four aromatic proton signals, δ_H 7.63 (brs, H-2′, 6′), 6.64 (s, H-3), 6.53 (d, H-8) and 6.18 (d, H-6) appeared (Table 3). The attachment of glucose to 3′,5′-positions of tricetin was determined by HMBC correlation between glucosyl anomeric proton signal at δ_H 4.90 (2H, d) and C-3′,5′ signal at δ_C 146.9–147.0 (Fig. S5). The β-pyranose form of the glucose was assigned by ¹H NMR and NOESY correlation (Fig. S6). Thus, 13 was identified as tricetin 3′,5′-di-O-glucopyranoside. The compound has been reported from Dacrycarpus dacrydioides (Podocarpaceae) (Markham and Whitehouse, 1984).

Compound 14 showed the peaks of deprotonated molecule at m/z 655 [M − H]⁻ and fragment ions at m/z 495 [M + H − 162]^་and 333 [M + H − 162×2]^་ on LC/MS, suggesting that 14 is monomethoxylated myricetin attached to 2 mol hexoses. UV spectral properties were the same with those of 4, that is, 14 is methoxylated and di-O-glycosylated at 3,3′,5′-positions. In ¹H NMR spectrum, four aromatic proton signals, δ_H 7.73 (d, H-2′), 7.65 (d, H-6′), 6.46 (d, H-8) and 6.20 (d, H-6) appeared (Table 2). The methoxy proton signal was observed at δ_H 3.94 (s). The attachment of methoxy group to 3′-position of myricetin was determined by HMBC correlation between methoxy proton signal at δ_H 3.94 and C-3′ carbon signal at δ_C 149.2 (Fig. S7 and S8). On the other hand, the attachment of hexose to 3,5′-positions of myricetin was determined by HMBC correlation between glucosyl anomeric protons at δ_H 5.36 and δ_H 4.89 and C-3 and C-5′ signals at δ_C 135.7 and and δ_C 146.6, respectively (Fig. S7). The β-pyranose structure of glucose was assigned by ¹H NMR and NOESY correlation (Fig. S8). Thus, 14 was identified as laricitrin 3,5′-di-O-glucopyranoside. The compound has been reported from the aerial parts of Medicago littoralis Rhode. (Fabaceae) (Alessandra et al., 2010).

The LC/MS analysis of 15 detected the peaks at m/z 465 [M + H]⁺ and 463 [M − H]⁻. UV spectral properties indicated that 15 is a flavone having orth-dihydroxyl group in the B-ring. Five aromatic proton signals, δ_H 7.48 (d, H-2′), 7.20 (d, H-6′), 6.59 (s, H-3), 6.49 (d, H-8) and 6.21 (d, H-6) appeared in the NMR spectrum (Table 3). The attachment of glucose to 3′-position of aglycone was determined by HMBC correlation between glucosyl anomeric proton signal at δ_H 4.87 and C-3′ signal at δ_C 147.8 (Fig. S9). The β-pyranose form of the glucose was assigned by ¹H NMR and NOESY correlation (Fig. S10). Thus, 15 was identified as tricetin 3′-O-glucopyranoside. The compound has been reported from the leaves of Thuja occidentalis L. and Lathyrus pratensis L. (Lamer et al., 1968; Reynauld et al., 1981)

Compound 16 showed the peaks at m/z 479 [M + H]⁺ and 477 [M − H]⁻ on LC/MS. UV spectral properties were identical with those of 13. In ¹H NMR spectrum, five aromatic proton signals, δ_H 7.60 (H-2′), 7.32 (H-6′), 6.68 (s, H-3), 6.52 (d, H-8) and 6.21 (d, H-6) occurred (Table 3). Methoxy proton signal was observed at δ_H 3.97 and correlated with C-3′ signal at δ_C 147.3 by HMBC, showing that a methoxy group is attached to 3′-position of the aglycone (Fig. S11). The glycosylation at 5′-position of the aglycone was determined by HMBC correlation between glucosyl anomeric proton at δ_H 4.90 and C-5′ signal at δ_C 104.6. The pyranose form of the sugar was assigned by NOESY correlation (Fig. S12). Thus, 16 was identified as selagin 5′-O-glucopyranoside, which is an undescribed flavone (Buckingham J, Munasinghe V R N, 2015).

Flavonoid comparison among three Puya species

HPLC chromatograms of the extracts from three Puya species are shown in Fig. 1 and Table 1. Flavones 8 (16.1%) and 9 (8.3%) were detected as the main components of P. coerulea var. violacea. In pale yellow P. chilensis, flavonols 11 (23.1%) and 14 (13.2%), and flavone 16 (36.3%) were detected as the main components. HPLC chromatogram showed that the turquoise flowers of P. alpestirs contain all of 8, 9, 11, 14 and 16; thus, P. alpestirs seems to have an intermediated characters between blue and pale-yellow Puya species.

The HPLC profiles of three Puya species were compared using a principal component (PC) analysis (Fig. 2). The proportion of variance was 0.6324 in first PC and 0.3079 in second PC. The 2-dimension scatter diagram of the PC analysis is shown in Fig. 2A. The content of anthocyanin 1 was the main factor having a positive effect on the first PC value. The second PC value was positively affected by 11 (myricetin 3,3′,5′-tri-O-glucoside), 14 (laricitrin 3,5′-di-O-glucoside) and 16 (selagin 5′-O-glucoside). The locus of P. coerulea var. violacea was closer to that of P. alpestris (distance = 0.366) than of P. chilensis (distance = 1.092).

In addition, the scatter diagram excluding anthocyanin-derived factors is shown in Fig. 2B. The proportion of variance was 0.4690 in the first PC and 0.3600 in the second PC. The first PC value was influenced positively by the contents of 11 (myricetin 3,3′,5′-tri-O-glucoside) and 16 (selagin 5′-O-glucoside), and negatively by those of 8 (luteolin 4′-O-glucoside) and 9 (apigenin 4′-O-glucoside). The distance between the loci of P. coerulea var. violacea and P. alpestris (0.970) and that of P. chiliensis and P. alpestris (0.965) were almost the same.

Aglycone analysis

The proportion of flavonol and flavone aglycones in the flower of three Chilean Puya species was examined by HPLC analysis of the products obtained by acid hydrolysis. Ten aglycones were detected in total (Fig. 3, Table S1). Among them, luteolin type flavones such as luteolin and chrysoeriol (37.5%), and tricetin type flavones (20.5%) including tricetin and selagin were the major flavonoids in P. coerulea var. violacea. In the extracts of P. chilensis, high proportions of tricetin (44.3%), and myricetin type flavonols, including myricetin and laricitrin (32.9%) were contained. In P. alpestris, luteolin type flavones (31.7%) including luteolin and chrysoeriol, and the myricetin type flavonols (23.7%) were contained as its major flavonoids. The proportion charts of the aglycones in respective species are shown in Fig. 3C, together with presumable flavonoid biosynthetic pathway (Fig. 3B).

Flower color analysis using absorption spectra

The visible absorption spectra of the blueish P. coerulea var. violacea, turquoise P. alpestris, and pale-yellowish P. chilensis are shown in Fig. 4A. The petal of P. alpestris exhibited the absorption maxima at 573, 614 and 678 nm, as reported in the previous report (Mizuno et al., 2021). Two absorption maxima were observed at 579 and 613 nm in the petal of P. coerulea var. violacea, indicating that the petal is blueish. On the other hand, absorption was mainly observed at 400–500 nm in the petal of P. chilensis. The spectral curve did not show any absorption peaks of carotenoids, whose absorption maxima are around ca. 400–500 nm (Zscheile et al., 1942). In addition, a small peak at 678 nm was observed in the petal of P. chilensis, which was presumed to be derived from chlorophylls (Mizuno et al., 2021).

In vitro reconstruction of flower colors

The pH values of the pressed petal juice of P. coerulea var. violacea and P. chiliensis were 5.2 and 6.2, respectively. The petal pH of P. alpestris is found to be 6.2 (Mizuno et al., 2021). The flower color was reconstructed with the characteristic pigments of three Puya species, 1, 8 and 11, and analyzed by reflectance spectra (Fig. 3B). The reconstruction was carried out as follows: the mixture of 0.07 mM 1 and 0.1 mM 8 (1 + 8) for P. coerulea var. violacea; the mixture of 0.07 mM 1, 0.1 mM 8 and 0.1 mM 11 (1 + 8 + 11) for P. alpestris; and 0.1mM 11 (11) for P. chiliensis in two different pH buffers, 5.0 and 7.0. Buffer solutions of 1 + 8 in both pH reflected wavelengths corresponding to blue and red regions and appeared violet blue. The buffer solution of 1 + 8 + 11 at pH 7.0 had a similar reflectance to 1 + 8; but the secondary peak was shifted to lower wavelength (around 500 nm) by addition of 11, being turquoise in color. However, turquoise color was not expressed in pH 5.0, being dullish purple. The buffer solution of 11 at pH 7.0 appeared yellowish color, and its reflectance curve between 300–400 nm was different from the solution of pH 5.0.

Using these reflectance spectra, the color loci of each solution in the visual model of ultraviolet-sensitive (UVS) and violet-sensitive (VS)-avian species were modeled (Figs. 5 and 6). In the UVS-avian color space, the color loci of the bluish solutions of 1 + 8 at pH 5.0 and 7.0 were closely located, and displayed no significant difference in values of chromatic contrast (ΔS), achromatic contrast (ΔL) and spectral purity between the two pH. On the other hand, the pH had a larger effect for 11 and 1 + 8 + 11, compared to 1 + 8; the higher pH largely increased ΔS and spectral purity and decreased ΔL. In the VS-avian tetrahedral color spaces (Fig. 6E), although all color parameters followed the same tendency with those in the UVS color space, the effect of pH was relatively small.

Three anthocyanins, seven flavonols and six flavones were isolated from the flowers of Chilean Puya species. The flavonoids in these species were composed of various aglycones, and many of them were glucosylated at the B-ring such as 3’,4’- and/or 5’-positions. Flavonoids including anthocyanins have been reported from the genus Puya (Scogin, 1985; Williams, 1978). In addition, delphinidin 3,3′,5′-tri-O-glucoside and myricetin 3,3′,5′-tri-O-glucoside have been isolated from the turquoise flowers of P. alpestris (Mizuno et al., 2021). Furthermore, various glycosides of myricetin, quercetin, laricitrin, isorhamnetin and apigenin have been detected by MS analysis of the meristem and leaves of P. chiliensis (Jiménez-Aspee, et al., 2018). Our data on the detail identification of flavonoids using NMR might contribute to the further chemotaxonomic, biological, and pharmaceutical study of the related species.

In this study, we showed the distribution of flavonoids in the flowers of three Chilean Puya species by HPLC (Fig. 1). From the result, we presumed that turquoise flowered P. alpestris contains both the compounds contained in blue flowered P. coerulea var. violacea and pale-yellow flowered P. chilensis. The relationship among three Puya species visualized in the scatter diagram by 2-dimension PC analysis (Fig. 2) showed that the presence of anthocyanin 1 significantly affected the first PC. The locus of P. alpestris was located closer to that of P. coerulea var. violacea than P. chilensis. In the PC analysis without anthocyanins, the first PC showed that P. alpestris is an intermediate position between P. coerulea var. violacea and P. chiliensis. Meanwhile, the second PC value of P. alpestris was notably higher than the other two species, which may be accounted by the abundance of 8, 9, and 11. It means that the flavonoid composition of P. alpestris tends to be intermediate between those of the other two species, but is characteristic.

Variation in the flower color is often associated with the flavonoid composition regulated through the biosynthesis pathway, which has been discussed in the ecological and evolutional contexts (Wheeler and Smith 2019; Wheeler et al., 2021). Our aglycone analysis unveiled that the three species share the same aglycones, likely synthesized via a flavonoid biosynthesis pathway proposed in Fig. 3; but, the proportion differed in the three Puya species. It means that the regulation in the biosynthesis of flavonoid causes the different coloration in these flowers. The inter-specific difference in the proportion of the myricetin and tricetin types implies that the biosynthetic genes of flavonoid 3′,5′-hydroxylase (F3′5′H) are one of the key providing the color variations.

Pale-yellow flower coloration due to flavonols is unusual (Hashimoto et al., 2008; Iwashina et al., 1988). It has been mainly known to be attributed by 6- or 8-hydroxtylated yellow flavonols, chelated with alum, or under relatively high pH conditions in vacuole (Mishio et al., 2006; Stewart et al., 1975; Harborne 1968; Tanikawa et al., 2008). The turquoise petal of P. alpestris and pale-yellow P. chiliensis had relatively high pH 6.2 and 6.0, respectively. On the other hand, the pH of bluish flowered P. coerulea var. violacea, was general pH 5.2. We propsed that relatively high vacuolar pH is adaptive in pale-yellow and turquoise flowers of Puya species.

It has been known that the subtle pH differences cause great changes in the absorption spectrum of flower pigments (Stavenga et al., 2021). In addition, it has been suggested that the floral spectral properties are tuned to optimize their visibility to pollinators as an advertisement (Stavenga et al., 2021; van der Kooi 2021). The visual modeling of the flower color reconstructed by pigments in vitro demonstrated the effect of pH-dependent color changes on avian visual perceptions (Figs. 4, 5 and 6). In both of UVS- and VS-avian color spaces, similar pH-depending changes were observed on ΔS and spectral purity values. By increasing pH from 5.0 to 7.0, the values largely increased in 11 and 1 + 8 + 11, especially in the UVS-avian color space. The effect of pH seemed relatively small for VS-birds. The high vacuolar pH in P. alpestris (pH 6.2) and P. chilensis (pH 6.2) would contribute to increase the floral ΔS and spectral purity, being conspicuous to UVS-birds rather than VS-birds. The most frequent visitors to P. alpestris and P. chilensis are the Chilean mockingbird, Mimus thenca (Hornung-Leoni et al., 2013), whose visions is considered to be UVS-type (Ödeen et al., 2011). The unusual high pH of P. alpestris and P. chilensis may be an adaptive trait to attract UVS-birds. On the other hand, no significant difference was detected in 1 + 8 between the two pH. In blue P. coerulea var. violacea (pH 5.2), to which the VS-type giant hummingbird Patagona gigas frequently visit (Hornung-Leoni et al., 2013), its color parameters ΔS, ΔL and spectral purity may not be affected by pH at least in the range of 5–7.

In this study, we compared the coloration at different pH using 1, 8 and 11, which have been shown as pigments responsible for the turquoise coloration in a previous study (Mizuno et al., 2021). Nevertheless, we isolated and identified 16 compounds from petals; there might be more complex relationship between flower color, pigments, and pH in nature. Reflectance spectral data of intact petals would be also of importance for understanding the color characteristics. We will conduct further studies.

Specialized metabolites attract pollinators as multiple stimuli including a visual cue, and their vast diversity has been shown (Harborne and Smith, 1978; Mori et al., 2023). The glycosylation and acylation of anthocyanins cause the modification of its absorption spectrum (Giusti et al., 1999; Mazza and Brouillard, 1987). Furthermore, the spectral shifts also can be occurred by the presence of accessory pigments (Mazza and Brouillard, 1990; Trouillas et al., 2016). We need to address this diversity to understand plant-pollinator interactions (Wong et al., 2023). In this study, we first applied the pollinator visual modeling for isolated pigments, which may be an effective tool for ecological and evolutionary studies of plant-pollinator interactions. The modeling of in vitro reconstructed color provides the additional perspective to chemical studies of flower pigments.

Phylogenetic studies of the Chilean Puya species have suggested that the turquoise flowered P. alpestris is a hybrid between the blue and pale-yellow Puya species (Jabaily and Sytsma, 2010; Jabaily and Sytsma, 2013). This study revealed that the chemical structures of flavonoids and their distribution in the different flower colored Chilean Puya species, i.e., blue P. coerulea var. violacea, turquis P. alpestris, and pale-yellow P. chilensis. The flavonoid composition of P. alpestris without anthocyanin is essentially intermediate between blue and pale-yellow flowered species, which is congruent with the molecular phylogeny. The visual modeling using reconstructed flower color with the isolated compounds suggested that relatively higher pH of P. alpestris and P. chiliensis flowers is an adaptive trait to their UVS-bird pollinators. We propose that the turquoise color expression of P. alpestris is caused by regulation of the pH and flavonoid composition. Further analysis might be needed for understanding of the variation of flower colors of Chilean Puya species.

Plant material

Fresh petals of Puya coerulea var. violacea (Brongn.) (16.0 g) were collected from the Tsukuba Botanical Garden, National Museum of Nature and Science (Tsukuba, Japan) in March 2019. Fresh petals of P. chilensis Molina (21.0 g) were collected from the Atagawa Banana Wani-en (Shizuoka, Japan) in March 2021. Fresh petals of P. alpestris (Poepp.) Gay (ca. 10 g) were collected from the Botanical Gardens of Osaka Metropolitan University (Osaka, Japan) in June 2019. Tip side of the petals was used for the measurement of UV–vis absorption spectra, HPLC analysis and pH measurement.

Isolation and identification of the anthocyanins, flavonols and flavones

Fresh petals of each species were extracted using 5% HCOOH in MeOH. After removing solvents by evaporation, each extract was applied to column chromatography using Diaion HP-20SS (Mitsubishi Chemical Co., Japan) and eluted with 0.5% TFA (trifluoroacetic acid), 10% MeCN containing 0.5% TFA and 25% MeCN containing 0.5% TFA. The fractions were chromatographed on a Sephadex LH-20 gel, eluenting with MeOH/H₂O/HCOOH (70:25:5). The mixtures were applied to preparative HPLC using a Shimadzu HPLC system equipped with an Inertsil ODS-4 column (I.D. 10 × 250 mm, GL Sciences Inc., Japan) under the following conditions. The eluent was 10% MeCN containing 5% HCOOH to 20% MeCN containing 5% HCOOH at a flow rate of 3 − 3.5 mL min^− 1.

Three anthocyanins (1–3), four flavonols (4–7), and three flavones (8–10) were isolated from P. coerulea var. violacea, and three flavonols (11, 12 and 14) and three flavones (13, 15 and 16) were from P. chilensis. They were characterized by UV–vis spectroscopy, wavelength at 400–700 nm for anthocyanins, and 220–500 nm for the other flavonoids (Mabry et al., 1970), liquid chromatography-mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR), and HPLC and TLC comparisons with authentic specimens. The origins of the authentic specimens were as follows: delphinidin 3,3′,5′-tri-O-glucoside, apigenin and luteolin 4′-O-glucosides, and myricetin 3-O-rutinoside-3′,5′-di-O-glucoside and 3,3′,5′-tri-O-glucoside from the flowers of P. alpestris (Mizuno et al., 2021), quercetin 3,3′-O-glucoside was obtain from partial hydrolysis of 4, delphinidin 3-O-glucoside and quercetin 3-O-rutinoside (rutin) from Funakoshi Co., Ltd. (Tokyo, Japan), and isorhamnetin 3-O-rutinoside from the leaves of Saussurea pricei N.D.Simpson (Asteraceae) (Kusano et al., 2010).

The MS was performed on a Shimadzu LC/MS system using an Inertsil ODS-4 column (I.D. 10×250 mm, GL Sciences Inc., Tokyo) equipped with a PDA detector (4.5 kV for ESI⁺ and 3.5 kV for ESI⁻; interface temperature as 250°C).

NMR spectroscopy was recorded on a Bruker AVANCE III HD 800 spectrometer equipped with a 5-mm TCI cryogenic probe and Z-axis gradient (Bruker Biospin AG, Switzerland). All spectra were obtained in 0.6 mL of the deuterated solvent placed inside 5-mm NMR tubes (Wilmad Lab Glass, USA) at 298 K. The compounds of 4 and 12–16 were dissolved in CD₃OD as the NMR solvent. Chemical shifts are reported in ppm and referenced to the residual methanol peaks observed at δ_H = 3.31 and δ_C = 49.00. Standard Bruker pulse sequences were employed. All NMR data reported were obtained via 1D ¹H and ¹³C NMR, 2D ¹H DQF-COSY, 2D ¹H NOESY (mixing time = 400, 500, or 750 ms), 2D ¹H{¹³C} edited-HSQC, 2D ¹H{¹³C} HMBC, and 2D ¹H{¹³C}-HMQC-COSY experiments. Data analyses were performed using Bruker TopSpin software (v. 3.5; Bruker Biospin AG, Switzerland).

HPLC comparisons

The fresh petals were extracted with MeOH containing 5% HCOOH. The extract was filtered through a 0.45 µm GL Chromatodisk filter 13N (GL Sciences Inc.) and then subjected to HPLC. HPLC analysis was performed on a Shimadzu HPLC system equipped with an SPD-20A UV-vis detector (Shimadzu) using a InertSustain C18 column (I.D. 4.6 × 250 mm, GL Sciences Inc.) at a flow rate of 1.0 mL min^− 1. The mobile phase consisted of solvent A (5% HCOOH aq.) and solvent B (90% MeCN containing 5% HCOOH). The gradient solution program was as follows: linear gradient from 10 to 50% B for 35 min, followed by 50% B for 10 min. Flavonoid composition was compared among the three species with LCSolution software (Shimadzu).

Aglycone analysis

Aglycone analysis was performed according to Mizuno et al. (2020). Crude extracts (1 mL) from the three Puya species were added into 0.1 mL 12% aq. HCl, and treated for 40 min at 100°C. After cooling, the solutions were diluted with 4 mL water and applied to Sep-pak C18 cartridge (Waters) eluted with 2 mL H₂O, 2 mL 10% MeCN containing 0.5% TFA and 1 mL MeOH. The MeOH fractions were applied to HPLC (See section 5.4.). Authentic myricetin was purchased from Sigma, and quercetin, luteolin, apigenin and chrysoeriol was from Extrasynthese (France). Authentic kaempferol and isorhamnetin were isolated from the fruits of Sophora japonica L. and Epiphyllum hybrida (Cactaceae), respectively (Iwashina and Kondo 1985). Authentic laricitrin, tricetin and selagin were obtained by acid hydrolysis of 14, 15 and 16.

Absorption spectra

UV–vis absorption spectra (400–700 nm) of the petals of three Puya species were measured according to Mizuno et al. (2021) using a UV-2600 spectrophotometer (Shimadzu, Japan) equipped with an integrated sphere ISR-2600 (Shimadzu).

Chemical data of the compounds

Delphinidin 3,3′,5′-tri-O-glucoside (1, Preternatin C5). TLC: R_f 0.03 (BAW), 0.02 (BuHCl), 0.38 (1% HCl), 0.57 (AHW); UV–vis: λ_max (nm) 0.01% HCl–MeOH 276, 522; E₄₄₀/E_max 32%; +AlCl₃ no change; LC-MS: m/z 789 [M]⁺, 627 [M − 162]⁺, 303 [M − 162×3]⁺.

Delphinidin 3′,5′-di-O-glucoside (2). TLC: R_f 0.08 (BAW), 0.02 (BuHCl), 0.07 (1% HCl), 0.29 (AHW); UV–vis: λ_max (nm) 0.01% HCl–MeOH 278, 532; E₄₄₀/E_max 27%; +AlCl₃ no change; LC-MS: m/z 627 [M]⁺, 303 [M − 162×2]⁺.

Delphinidin 3-O-glucoside (3). TLC: R_f 0.13 (BAW), 0.02 (BuHCl), 0.03 (1% HCl), 0.10 (AHW); UV–vis: λ_max (nm) 0.01% HCl–MeOH 278, 543; E₄₄₀/E_max 21%; +AlCl₃ bathochromic shift; LC-MS: m/z 465 [M]⁺, 303 [M − 162]⁺.

Quercetin 3-O-rutinoside-3′-O-glucoside (4). TLC: R_f 0.47 (BAW), 0.64 (15% HOAc), 0.58 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow; UV: λ_max (nm) MeOH 266, 349; +NaOMe 276, 324, 398 (inc.); +AlCl₃ 274, 302, 354, 393; +AlCl₃/HCl 275, 302, 349, 393; +NaOAc 273, 328, 399; +NaOAc/H₃BO₃ 267, 354; LC-MS: m/z 627 [M + H − 146]⁺, 303 [M + H − 162×2 − 146]⁺, 771 [M − H]⁻. ¹H and ¹³C NMR data, see Table 2.

Quercetin 3,3′-di-O-glucoside (5). TLC: R_f 0.59 (BAW), 0.50 (15% HOAc), 0.68 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow. UV: λ_max (nm) MeOH 266, 346; +NaOMe 278, 324, 402 (inc.); +AlCl₃ 273, 303, 347, 390; +AlCl₃/HCl 273, 305, 352, 402; +NaOAc 272, 316, 397; +NaOAc/H₃BO₃ 267, 353; LC-MS: m/z 625 [M − H]⁻, 463 [M − H−162]⁻.

Quercetin 3-O-rutinoside (6, rutin). TLC: R_f 0.69 (BAW), 0.49 (15% HOAc), 0.72 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow. UV: λ_max (nm) MeOH 259, 358; +NaOMe 276, 324, 413 (inc.); +AlCl₃ 274, 305, 325, 426; +AlCl₃/HCl 269, 299, 356, 396; +NaOAc 272, 306, 398; +NaOAc/H₃BO₃ 262, 318, 379; LC-MS: m/z 303 [M + H − 162 − 146]⁺, 609 [M − H]⁻.

Isorhamnetin 3-O-rutinoside (7, narcissin). TLC: R_f 0.78 (BAW), 0.55 (15% HOAc), 0.84 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: dark yellow. UV: λ_max (nm) MeOH 267, 350; +NaOMe 270, 329, 399 (inc.); +AlCl₃ 266, 296, 353; +AlCl₃/HCl 270, 298, 353; +NaOAc 268, 353; +NaOAc/H₃BO₃ 267, 352; LC-MS: m/z 317 [M + H − 162 − 146]⁺, 623 [M − H]⁻.

Luteolin 4′-O-glucoside (8). TLC: R_f 0.73 (BAW), 0.11 (15% HOAc), 0.79 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: dark purple. UV: λ_max (nm) MeOH 269, 336; +NaOMe 265, 296, 368 (dec.); +AlCl₃ 275, 293, 341, 387sh ; +AlCl₃/HCl 279, 296, 343, 387sh; +NaOAc 269, 337; +NaOAc/H₃BO₃ 270, 337; LC-MS: m/z 449 [M + H]⁺, 287 [M + H − 162]⁺, 447 [M − H]⁻.

Apigenin 4′-O-glucoside (9). TLC: R_f 0.81 (BAW), 0.19 (15% HOAc), 0.81 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: dark purple. UV: λ_max (nm) MeOH 270, 326; +NaOMe 289, 354 (dec.); +AlCl₃ 291, 334, 386sh; +AlCl₃/HCl 291, 334, 375sh; +NaOAc 270, 325; +NaOAc/H₃BO₃ 270, 327; LC-MS: m/z 433 [M + H]⁺, 271 [M + H − 162]⁺, 431 [M − H]−.

Tricetin 4′-O-glucoside (10). TLC: R_f 0.74 (BAW), 0.12 (15% HOAc), 0.77 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: dark purple. UV: λ_max (nm) MeOH 271, 331; +NaOMe 274, 383 (dec.); +AlCl₃ 279, 335, 390sh; +AlCl₃/HCl 279, 300, 335, 387sh; +NaOAc 271, 333; +NaOAc/H₃BO₃ 271, 333; LC-MS: m/z 463 [M − H]⁻, 301 [M − H−162]⁻.

Myricetin 3,3′,5′-tri-O-glucoside (11). TLC: R_f 0.26 (BAW), 0.58 (15% HOAc), 0.37 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow. UV: λ_max (nm) MeOH 248, 267, 350; +NaOMe 278, 330, 400 (inc.); +AlCl₃ 275, 305sh, 354, 399; +AlCl₃/HCl 276, 305sh, 349, 396; +NaOAc 266, 322sh, 407; +NaOAc/H₃BO₃ 266, 301sh, 362; LC-MS: m/z 643 [M + H − 162]⁺, 481 [M + H − 162×2]⁺, 319 [M + H − 162×3]⁺, 803 [M − H]⁻.

Myricetin 3,3′-di-O-glucoside (12). TLC: R_f 0.40 (BAW), 0.32 (15% HOAc), 0.52 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow. UV: λ_max (nm) MeOH 254, 297, 356; +NaOMe 275, 328, 412 (inc.); +AlCl₃ 273, 306, 425; +AlCl₃/HCl 273, 306, 357, 400; +NaOAc 269, 327, 410; +NaOAc/H₃BO₃ 260, 306, 378; LC-MS: m/z 481 [M + H − 162]⁺, 319 [M + H − 162×2]⁺,641 [M − H]−. ¹H and ¹³C NMR data, see Table 2.

Tricetin 3′,5′-di-O-glucoside (13). TLC: R_f 0.29 (BAW), 0.10 (15% HOAc), 0.37 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow. UV: λ_max (nm) MeOH 269, 340; +NaOMe 258, 330, 399 (inc.); +AlCl₃ 278, 299, 352, 385; +AlCl₃/HCl 279, 298, 347, 382; +NaOAc 259, 401; +NaOAc/H₃BO₃ 267, 354, 398; LC-MS: m/z 627 [M + H]⁺, 625 [M − H]⁻, 311 [M − H−162×2]⁻. ¹H and ¹³C NMR data, see Table 3.

Laricitrin 3,5′-di-O-glucoside (14). TLC: R_f 0.37 (BAW), 0.34 (15% HOAc), 0.59 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow. UV: λ_max (nm) MeOH 256, 267, 348; +NaOMe 275, 331, 401 (inc.); +AlCl₃ 274, 304, 358, 398; +AlCl₃/HCl 276, 300, 349, 393; +NaOAc 267, 360; +NaOAc/H₃BO₃ 265, 328, 408; LC-MS: m/z 495 [M + H − 162]⁺, 333 [M + H − 162×2]⁺, 655 [M − H]⁻; ¹H and ¹³C NMR data, see Table 2.

Tricetin 3′-O-glucoside (15). TLC: R_f 0.38 (BAW), 0.17 (15% HOAc), 0.56 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow. UV: λ_max (nm) MeOH 250, 265, 351; +NaOMe 271, 326, 407 (inc.); +AlCl₃ 271, 307, 356, 388; +AlCl₃/HCl 274, 423; +NaOAc 265, 330sh, 403; +NaOAc/H₃BO₃ 258, 374; LC-MS: m/z 465 [M + H]⁺, 463 [M − H]⁻; ¹H and ¹³C NMR data, see Table 3.

Selagin 5′-O-glucoside (16). TLC: R_f 0.47 (BAW), 0.21 (15% HOAc), 0.49 (BEW); Color under UV (365 nm): dark purple, UV/NH₃: yellow. UV: λ_max (nm) MeOH 269, 336; +NaOMe 275, 389 (inc.); +AlCl₃ 275, 305, 358, 392; +AlCl₃/HCl 278, 300, 348, 393; +NaOAc 262, 322; +NaOAc/H₃BO₃ 266, 348, 404; LC-MS: m/z 479 [M + H]⁺, 477 [M − H]⁻; ¹H and ¹³C NMR data, see Table 3.

Principal component (PC) analysis

The PC analysis was performed with the R (R Core Team, 2021) using HPLC chromatograms of the three Puya species. A total of 20 variables including flavones, flavonols and anthocyanins (Table 1), 17 variables including flavones and flavonols (Table 1), and ten variables in aglycone analysis were used (Table S1).

Visual model of pollinators and color variables using the isolated compound

The petal tips of two Puya species, P. coerulea var. violacea and P. chilensis, were homogenized to obtain the pressed juice. pH of the juice was immediately measured on three independently pressed juice for each species, using a twin pH meter AS-212 (HORIBA, Japan). The pH in the petal tips of P. alpestris refers to our previous study (Mizuno et al., 2021).

The flower color of the three Puya species was modeled and reconstructed with 0.07 mM 1 + 0.1 mM 8 (1 + 8) for P. coerulea var. violacea, 0.07 mM 1 + 0.1 mM 8 + 0.1 mM 11 (1 + 8 + 11) for P. alpestris, and 0.1 mM 11 (11) for P. chilensis. Each solution was prepared at pH 5.0 and 7.0 in Mcilvaine buffer. The reflectance spectra (300–750 nm) of the solutions were measured on a UV-2600 spectrophotometer (Shimadzu) equipped with an integrated sphere ISR-2600 (Shimadzu) using a D30 cell, divided by center partition (GL Sciences Inc., Japan). Ba₂SO₄ powder was used as a white standard.

The color loci of each solution in the pollinator’s perceptual color space were computed based on their diffuse reflectance spectra with the R v. 4.1.2 (R Core Team, 2021) package pavo v. 2.7.0 (Maia et al., 2019). To model the vision of avian pollinators, the color loci were projected in the tetrahedron vision space (Endler and Mielke, 2005). Since floral visitors of P. coerulea, P. alpestris and P. chilensis (Hornung-Leoni et al., 2013) include both UVS- and VS-type (Ödeen and Håstad, 2010), the calculations were performed with the spectral sensitivity of both UVS- and VS-avian (Maia et al., 2019). The CIE D65 daylight irradiance spectrum was used as a model of daylight irradiance. Averaged reflectance spectrum of leaves in pavo was assumed as the background.

The distance from the flower locus to the tetrahedron achromatic center was calculated as spectral purity (Stoddard and Prum, 2011). Achromatic (ΔL) and chromatic contrasts (ΔS) against a leaf background were calculated in the receptor noise-limited model (RNL model; Vorobyev and Osorio, 1998). ΔL was calculated based on the double cone responsible for chromatic processing, according to Siddiqi et al. (2004). ΔS was obtained by weighting the Euclidean distance of the photoreceptor quantum catches by the Weber fraction of the cones. The Weber fraction (ω) was assumed to be 0.1 in the LWS mechanism, based on the empirically estimated value from the Pekin robin (Leiothrix lutea) (Maier, 1992; Olsson et al., 2018). Photoreceptor abundance of the cone types was estimated to be 1:2:2:4 for the UVS:SWS:MWS:LWS, according to those of L. lutea (Vorobyev and Osorio, 1998). ΔS was expressed in just noticeable difference (JND), where 1 JND is the theoretical threshold of color discrimination for stimuli viewed simultaneously under ideal viewing conditions (Vorobyev et al., 2001).

Statistical analyses were performed with R v. 4.1.2 (R Core Team, 2021). Significant differences among species were tested by one-way analyses of variance (ANOVA), using the multcomp package. The ANOVA was followed by a Tukey–Kramer post-hoc analysis to test the pairwise comparisons. p < 0.05 was considered statistically significant.

Acknowledgements

The authors are grateful to Hideo Shimizu, Atagawa Tropical & Alligator Garden, Japan, Dr. Chie Tsutsumi, Tsukuba Botanical Garden, National Museum of Nature and Science, Japan, and some staffs for offering the flowers of living collections. The authors are grateful to Professor, Dr. Naonobu Noda, National Agriculture and Food Research Organization, Tsukuba-City, Ibaraki, Japan for useful advice. This work was supported by JSPS KAKENHI Grant Number 22K05643.

Competing interests: No competing interests exist

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Tables 1 to 3 are available in the Supplementary Files section.

TableS1.PuyaJPRrvar3.xlsx
Table S1. The proportion of the flavone and flavonol aglycones from the flower of three Puyaspecies.
FigSup.PuyaJPRver3.pptx
Table1.PuyaJPRrvar3.xlsx
Table. 1. HPLC profiles from the flowers of three Chilian Puya species
Table2.PuyaJPRrvar3.xlsx
Table. 2. ¹H (800MHz) and ¹³C (200 MHz) NMR data of three flavonols (4, 12 and 15) from the flower of Puya species.
Table3.PuyaJPRrvar3.xlsx
Table 3. ¹H (800 MHz) and ¹³C (200 MHz) NMR data of three flavones (13, 14 and 16) from the flower of Puya species.

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published 04 Mar, 2024

Read the published version in Journal of Plant Research →

Editorial decision: Minor revision
04 Jan, 2024
Reviewers agreed at journal
25 Nov, 2023
Reviewers invited by journal
13 Nov, 2023
Editor assigned by journal
09 Nov, 2023
First submitted to journal
08 Nov, 2023

You are reading this latest preprint version

Floral pigments and their perception by avian pollinators in three Chilean Puya species

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Flavonoid comparison among three Puya species

Aglycone analysis

Flower color analysis using absorption spectra

In vitro reconstruction of flower colors

Discussion

Conclusion

Experimental

Plant material

Isolation and identification of the anthocyanins, flavonols and flavones

HPLC comparisons

Aglycone analysis

Absorption spectra

Chemical data of the compounds

Principal component (PC) analysis

Visual model of pollinators and color variables using the isolated compound

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1