Convergent evolution of a blood-red nectar pigment in vertebrate-pollinated flowers

Abstract

Nearly 90% of flowering plants depend on animals for reproduction. One of the main rewards plants offer to pollinators for visitation is nectar. Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here, we show that the nectar's red color is derived from a previously undescribed alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including a carbonic anhydrase that increases nectar pH, an aryl-alcohol oxidase that produces a pigment precursor, and a ferritin-like catalase that protects the pigment from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a pigment with a stable imine bond. We subsequently verified that synthetic nesocodin is indeed attractive to Phelsuma geckos, the most likely pollinators of Nesocodon We also identify nesocodin in the red nectar of the distantly related and hummingbird-visited Jaltomata herrerae and provide molecular evidence for convergent evolution of this trait. This work cumulatively identifies a convergently evolved trait in two vertebrate-pollinated species, suggesting that the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this type of adaptation.

Keywords: Jaltomata; Nesocodon; gecko; nectar; nectaries.

Fig. 1.

The color and pH of N. mauritianus nectar change over time. (A and B) Newly opened flower (0 h); B is a close-up of A showing the color of five distinct nectar droplets (arrowhead). (C and D) Flower ∼24 h after opening; D is a close-up of C. (E) UV/visible absorbance spectra of nectars collected at 0 h (yellow), +8 h (orange), and +24 h (red) after dilution at 1:50 in deionized H₂O. Images of each undiluted nectar and pH are noted in the Inset. Structures of the pigments contributing to the absorbance peaks were initially identified by LC-MS (SI Appendix, Figs. S3, S4, and S9) and confirmed by multiple approaches (SI Appendix, Figs. S7, S8, S10–S14). (F) pH-dependent formation of colored products derived from 1 mM sinapaldehyde and 10 mM proline in 25 mM buffers of varying pH, including 6.0 (MES), 6.5 (MES), 7.0 (HEPES), 7.5 (HEPES), 8.0 (HEPES), and 8.5 (Tricine). (G) Color of 1 mM sinapaldehyde in 50 mM Na(H)CO₃ (pH 9.0), with and without 10 mM l-proline after a 30-min incubation at 21 °C.

Fig. 2.

A carbonic anhydrase is responsible for nectar alkalinization. (A) SDS-PAGE analysis of 0 h, +8 h, and +24 h nectars (18 μL each; see SI Appendix, Fig. S17 for protein identification). (B) In-gel carbonic anhydrase assay of 18 μL of +24 h nectar and a positive control without (Left) and with (Right) 1 mM acetazolamide added. (C) UV/visible absorbance (Abs) spectra and pH of nectar droplets treated in situ with carbonic anhydrase inhibitors. The carbonic anhydrase inhibitors sulfanilamide (SA) or acetazolamide (AZ) were added to separate 0-h nectar droplets in the same flower to a final concentration of 1 mM; a mock treatment containing an equal volume of 10% DMSO was included; one untreated nectar droplet was collected at 0 h and stored at 4 °C in the dark; and the treated nectar droplets were subsequently collected at +10 h, measured for pH, diluted 1:50 in H₂O, and evaluated by UV-Vis spectrophotometry. (D) Absorbance spectra of the same nectars from panel C after diluting 1:50 with 50 mM Tricine (pH 8.5). Additional representative results from individual treated flowers are shown in SI Appendix, Fig. S20.

Fig. 3.

NmNec3 can oxidize sinapyl alcohol into sinapaldehyde to serve as a precursor to nesocodin. (A) Purified NmNec3 (Inset) has sinapyl alcohol oxidase activity and can produce sinapaldehyde. The absorbance spectrum shown by the solid yellow line is the result of a reaction mix containing 1 mM sinapyl alcohol and 0.1 μg/mL NmNec3 in 50 mM Na(H)CO₃ (pH 9.0). Negative controls with sinapyl alcohol but no NmNec3 did not yield any sinapaldehyde (gray line); a 0.5 mM sinapaldehyde standard in 50 mM Na(H)CO₃ (pH 9.0) was used as a reference (dotted yellow line). (B) The same reactions as in A (with NmNec3), but also containing 10 mM proline produced a red-colored product (solid red line) consistent with both synthetic nesocodin (black dotted line; 1 mM sinapaldehyde plus 10 mM proline) and raw nectar (red dashed line; diluted 1:20). (C) Representative images showing the colors of the products of panels A and B. (D) Reaction sequence between sinapyl alcohol, NmNec3, and proline to yield nesocodin.

Fig. 4.

NmNec2 has catalase activity that can protect nesocodin from H₂O₂-mediated degradation. (A) Left, SDS-PAGE (4 to 20%) analysis of 18 μL of raw nonboiled and boiled nectar stained with Coomassie blue; Right, in-gel catalase assay after SDS-PAGE without sample boiling or addition of reducing agent. Arrowheads indicate location of NmNec3 and associated activity band. (B) Catalase activity of total nonboiled (orange line) or boiled (gray) nectar proteins. Assays contained 25 mM H₂O₂ in 50 mM HEPES (pH 8.0) and either 0.17 μg/μL of total nectar proteins (either boiled or nonboiled) or an equivalent volume of HEPES buffer as a negative control. (C) Catalase-mediated protection of natural nesocodin. Filtered (deproteinated) nectar was incubated with 10 mM H₂O₂ for 18 h with either total nonboiled (yellow line) or boiled (blue) nectar proteins (0.1 μg/mL final). Controls included the addition of heme-based catalase (green), no protein (gray), and no H₂O₂ added [orange, “+HEPES” (25 mM, pH 8.0)]. (D) Catalase-mediated protection of quasisynthetic nesocodin. All reactions contained 1 mM sinapyl alcohol, 10 mM proline, and 0.1 μg/μL NmNec3 in 50 mM Na(H)CO₃ (pH 9.0). A subset of reactions contained either 0.2 μg/μL total nectar protein or 0.1 μg/μL of heme-based catalase. Traces of absorbance spectra from three individual replicates are presented as labeled.

Fig. 5.

Nectars containing nesocodin are visible and conspicuous to pollinators (diurnal geckos and birds). (A) Reflectance of N. mauritianus and J. herrerae nectars and the surrounding petals. Shaded areas around the curves represent 1 SD. (B) Tetraplot showing N. mauritianus reflectance data from A projected onto the visual space of diurnal Phelsuma geckos. The vertices of the tetrahedron correspond to four different photoreceptors. (C) Achromatic contrast (gray points) and chromatic contrast (orange points) between nectar and the adjacent petals (N-P) and petals and the natural rocky background (P-BKG) for N. mauritianus. (D) Tetraplot showing J. herrerae reflectance data from A projected onto the visual space of its respective pollinator, the green-backed fire crown hummingbird S. sephaniodes. (E) Achromatic contrast (gray points) and chromatic contrast (red points) between N-F and P-BKG for J. herrerae. Contrasts are expressed in units of JND (just noticeable differences), and the higher the value, the more conspicuous the color should appear to the pollinators. Error bars are 1 SE.

Fig. 6.

Nectars containing nesocodin are attractive to diurnal geckos (Phelsuma). (A) Number of investigations made by geckos (n = 15) to tubes containing synthetic nectars with or without 3 mM nesocodin. Sources of nectar with nesocodin were visited significantly more often than sources without nesocodin (Wilcoxon signed-ranks test: W = 60, z = 2.65, two-tailed P = 0.008). (B) The net volume of nectar removed by geckos (n = 15) relative to evaporative control nectars in adjacent terraria without geckos. Significantly more of the nectar with nesocodin was consumed by the geckos (Wilcoxon signed-ranks test: W = 77, z = 2.4, two-tailed P = 0.0164). The experimental setup used for this study is graphically illustrated in SI Appendix, Fig. S27.

Fig. 7.

J. herrerae nectar contains nesocodin and analogous enzymes for its production. (A) Absorbance spectra of J. herrerae, J. procumbens, and N. mauritianus nectars. Inset: J. herrerae and J. procumbens flowers and nectars. (Note: the spectra of J. herrereae and N. mauritianus are from samples diluted 1:50, whereas that of J. procumbens is not diluted.) (B) SDS-PAGE analysis of nectar proteins (Left panels) from J. herrerae and J. procumbens and in-gel carbonic anhydrase activity assays (Right). The arrowheads indicate the locations of an α-carbonic anhydrase (α-CA) and sinapyl alcohol oxidase (SAO). (C) Sinapyl alcohol oxidase activity in Jaltomata nectars. Images of sinapyl alcohol and purified sinapyl alcohol oxidase (SAO; JhNec7) from J. herrerae are shown in the left two tubes. The right two tubes show the production of yellow sinapaldehyde after mixing sinapyl alcohol with either purified enzyme from J. herrerae or raw nectar from J. procumbens (note: J. procumbens produces too little nectar to allow for purification of the enzyme prior to the assay).