DOI: 10.5061/dryad.pc866t1kp
title: Data from: Florivory defence: are phenolic compounds distributed differently within perianths?

description: Plants might allocate chemical defenses unequally within attractive units
 of flowers including petals, sepals, and bracts because of variations in
 the probability of florivory. Based on the optimal defense theory, which
 predicts that plants allocate higher chemical defenses to tissues with
 higher probabilities of herbivore attack, we predicted that distal parts
 and sepals would have higher chemical defense allocations than proximal
 parts and petals. To test this prediction, we compared total phenolics and
 condensed tannins concentrations as well as presence of florivory within
 attractive units of ten angiosperm species. In agreement with the
 prediction, the overall results showed that the distal parts had higher
 total phenolics and condensed tannins than the proximal parts. On the
 other hand, contrary to the prediction, petals and sepals showed no
 tissue-specific variations. Florivory was more severe on the distal parts
 than the proximal parts, although statistical support for the variation
 was slightly weak, while the variations in presence of florivory between
 petals and sepals differed between the distal and proximal parts. These
 results may support the prediction of the optimal defense theory because
 distal parts of attractive units had higher presence of florivory and
 concentration of chemical defenses.
description: <strong>Studied species</strong>
 We selected ten perennial species (Table 1) whose flowers are
 mono-colored or have a uniform pattern along their attractive units, based
 on visual inspection. We selected these species to obtain sufficient
 samples for chemical measurements and to minimize the effects of color
 pattern on distributions of chemical compounds along attractive units. One
 exceptional species (Houttuynia cordata) having no perianth but white
 showy petaloid bracts was also used. For species belonging to Asteraceae
 (Aster ageratoides, Aster microcephalus and Helianthus tuberosus), we used
 only ray flowers because dissecting small tubular flowers was impractical.
 Aquilegia buergeriana has two floral phenotypes (purple and yellow) and we
 measured the chemical defenses and florivory separately for each
 phenotype. <strong>Study
 sites</strong> We collected samples from twelve
 locations in Miyagi, Iwate, and Aomori prefectures in northern Japan
 (Table 1). Aobayama, Aramaki, and Kawauchi are suburbs of Sendai city,
 Miyagi Prefecture, where we sampled flowers growing in natural vegetation
 facing paved or unpaved roads. Kagitoriyama and Izumigatake are
 mountainous regions in Miyagi Prefecture, where we collected samples
 growing in deciduous-coniferous mixed forests. Koeji and Nakanose are also
 suburbs of Sendai city, where we collected samples growing in natural
 vegetation along the Hirose river. Tsuta, Tashiro, and Hakkoda are
 mountainous regions in Aomori Prefecture, where we sampled flowers growing
 in damp vegetation in Tsuta, in dry vegetation in Tashiro, and in natural
 vegetation in the Botanical Garden of Tohoku University in Hakkoda.
 Hayasaka and Sodeyama are plateau grassland areas in Iwate Prefecture,
 where we sampled flowers growing in natural vegetation.
 <strong>Sample collection</strong>
 We sampled non-damaged flowers without apparent color changes
 caused by aging for chemical measurement during June to September 2016.
 For the chemical trait measurements, a minimum amount of dry mass was
 required. Although measuring the chemical traits for each individual would
 be ideal, for many species it was not possible to obtain sufficiently
 large samples from single individuals. Therefore, to keep some natural
 variation in the chemical traits among individuals and simultaneously
 fulfill the minimum requirement for the chemical measurements, we made
 sample groups consisting of several individuals. Because the number of
 flowers needed for one measurement depended on the flower size, we
 collected a suitable number of flowers for each group for each species
 (Table 1). In each location, we randomly selected patches of several
 individuals so that we could treat flowers collected from one patch as one
 sample group. We collected flowers for each sample group from individuals
 growing close to each other. All procedures after collection of flowers
 were applied to each sample group so that flowers from different groups
 were never mixed together. We cut flowers at their pedicels or peduncles
 and returned them to the laboratory as soon as possible.
 In the laboratory, we separated attractive units, i.e., petal,
 sepal, and bract samples into several parts using scissors or razors to
 compare the differences in chemical concentrations among them. Because of
 the diversity of floral morphology, we couldn’t apply a single
 segmentation method to all species. Therefore, we applied simple
 anatomical segmentations for most species but applied specific
 segmentation methods for some species. The list of segmentation methods of
 attractive units of flowers for each species and assignment of categories
 [i.e., position (distal or proximal) and part (petal or sepal)] for each
 segmented attractive unit is shown in Table 2. For
 species belonging to Asteraceae (A. ageratoides, A. microcephalus, H.
 tuberosus) and H. cordata, we divided each petal and bract into the
 proximal and distal parts at the center. For the species having colored
 sepals that attract pollinators (L. auratum, and A. buergeriana), we
 divided proximal and distal parts of petals and sepals separately by
 applying the segmentation rule above. Spurs of A. buergeriana were
 separated for measurement of chemical traits and treated as proximal parts
 of petals. Because H. sieboldii has corollas consisting
 of a narrow tubular section, an expanding section, and a lobe section, we
 divided them into these three parts for measurement of chemical traits.
 For statistical analyses, we treated the former two as the proximal parts
 and the lattermost as distal parts. Aconitum japonicum
 has zygomorphic flowers consisting of petals with spurs and three types of
 decorative sepals (lower sepals, lateral sepals, and a helmet) covering
 petals. Hence, we separated them into 1) petals with spurs, 2) lower
 sepals, 3) lateral sepals, and 4) helmet. The three types of sepals were
 also separated into distal and proximal parts by the same rule noted
 above. Because the petals including spurs of A. japonicum were quite
 small, the need to obtain sufficient sample for the chemical measurements
 precluded separating them into distal and proximal parts. Thus, the
 chemical traits were measured for seven groups for this species.
 Cardiocrinum cordatum sets slightly warped actinomorphic flowers
 horizontally. Each flower has three petals and sepals. The upper side of
 each flower has two sepals and one petal, while the lower side has one
 sepal and two petals. Therefore, we measured the chemical traits for the
 upper and lower petals and sepals separately. Also, because flowers of the
 species seemed to consist of a narrow tubular section, an expanding
 section, and a lobe-like section similar to H. sieboldii, we separated the
 petals and sepals into these three parts. For the statistical analyses, we
 treated the tubular and expanding sections as proximal and the lobe-like
 section as distal. Flowers of H. fulva have inner and
 outer petals, so we separated the attractive parts of each flower into
 inner petals, outer petals, and sepals. Each of these three attractive
 parts was further divided into distal and proximal parts.
 During sample dissection, we removed any pollen attached to
 floral samples by brushing gently. For the species having spurs (A.
 japonicum and A. buergeriana), we also removed nectar from them. We dried
 the separated samples with silica gel and kept them at room temperature in
 the silica gel until they were used for the chemical
 measurements. <strong>Chemical trait
 measurement</strong> Before the chemical
 measurements, we ground dried samples of a segmented group into powder
 using Micro SmashTM MS-100R (TOMY DIGITAL BIOLOGY CO., LTD., Tokyo,
 Japan). Following the Folin-Ciocalteu method (Julkunen-Tiitto, 1985), we
 measured total phenolics concentration using tannic acid (Wako Pure
 Chemical Industries, Ltd., Osaka, Japan) as the standard. We followed the
 butanol-hydrochloric acid-iron assay to measure condensed tannin
 concentration using commercially available cyanidin chloride as the
 standard (Makkar et al., 1999). The concentrations of total phenolics and
 condensed tannins were calculated from the absorbance values obtained
 using a Multiskan GO spectrophotometer (Thermo Fisher Scientific, Vantaa,
 Finland). To control for possible effects of differences in conditions
 among the measurement batches (i.e., sets of samples measured
 simultaneously), we measured all flower parts from the same sample group
 in one measurement batch and measured only one sample group in one batch
 for each species so that the fluctuation among measurement batches
 similarly affects all flower parts. In addition, our preliminary
 measurement showed that measurements with different sample weights
 resulted in slightly different measured values even for the same sample
 (Fig. 1). To reduce the effects of sample weight variation, we equalized
 the sample weight of each part to 15 mg, the standard weight of a powdered
 sample needed for the measurements, when possible. However, because some
 separated parts of some sample groups lacked the required weight, we
 equalized the sample weight of each part to the minimal weight among the
 parts in the same sample group. Because the dry weights were too light to
 equalize, we omitted the distal part of H. sieboldii in three sample
 groups. Also, because the weights of distal and proximal samples of
 lateral sepals in eight sample groups of A. japonicum were less than 5 mg,
 the chemical traits were measured using mixed samples of distal and
 proximal parts to avoid inaccurate measurement due to the measurement
 limit of the spectrometer. <strong>Measurements
 for presence of florivory</strong> It was
 impractical to precisely measure the amount of florivory for each part of
 attractive units for species in Asteraceae because they have many small
 ray flowers and in Ranunculaceae because they have complex floral
 structures. To apply the same method to all species and all parts of
 attractive units, we measured florivory as presence/absence. During the
 sample collections for the chemical measurements, we measured presence of
 florivory for randomly selected flowers. We recorded signs (i.e., missing
 parts on petals, sepals, and bracts) of florivory damage for proximal and
 distal parts of petals/sepals/bracts of each sampled flower in a binary
 manner. After the measurements, non-damaged flowers without apparent color
 changes due to aging were included in the chemical measurement
 samples.
 <strong>References</strong>
 Julkunen-Tiitto R. 1985. Phenolic constituents in the leaves of
 northern willows - methods for the analysis of certain phenolics. Journal
 of Agricultural and Food Chemistry 33: 213-217.<br> Makkar HPS,
 Gamble G, Becker K. 1999. Limitation of the butanol-hydrochloric acid-iron
 assay for bound condensed tannins. Food Chemistry 66: 129-133.
 <strong>Figure legend</strong>
 <strong>Fig. 1</strong>: Relationship between
 measured sample weight and concentration of total phenolics in Lilium
 auratum shown by the preliminary measurements. Symbols connected by a
 dashed line represent same sample measured with different weights. The
 figure file is included in the methods directory of the dataset.
 <strong>Tables</strong>
 <strong>Table 1</strong>: Detailed information of the
 species and sampling sites. Numbers of flowers and sample groups for each
 sampling site are also shown. 
 Family Species
 Size of one flower or flower head
 Segmentation Site name
 Location 
 Asparagaceae Hosta sieboldii
 (Paxton) J.W.Ingram var. sieboldii f. spathulata (Miq.)
 W.G.Schmid 4–5 cm
 Distal/Proximal Aramaki
 38°15'27.7"N 140°50'32.4"E
    
    
 Izumigatake 38°23'20.5"N
 140°43'09.7"E 
 Asphodelaceae Hemerocallis fulva L. var.
 kwanso Regel 10 cm
 Distal/Proximal of Petal/Sepal
 Aobayama 38°15'31.9"N
 140°51'21.0"E 
 Asteraceae Aster ageratoides Turcz. var.
 ageratoides 1.5–2 cm
 Distal/Proximal Kagitoriyama
 38°14'24.4"N 140°49'50.1"E
 Asteraceae Aster
 microcephalus (Miq.) Franch. et Sav. var. ovatus (Franch. et Sav.) Soejima
 et Mot.Ito 2.5 cm
 Distal/Proximal Kawauchi
 38°15'37.6"N 140°50'57.4"E
    
     Tsuta
 40°35'52.4"N 140°57'16.4"E
 Asteraceae
 Helianthus tuberosus L. 5–10 cm
 Distal/Proximal Koeji
 38°14'45.8"N 140°52'49.3"E
    
    
 Nakanose 38°15'38.4"N
 140°51'30.4"E 
 Liliaceae Cardiocrinum cordatum (Thunb.)
 Makino 12–18 cm Distal/Proximal
 of Petal/Sepal Aobayama
 38°15'17.7"N 140°51'23.4"E
 Liliaceae Lilium
 auratum Lindl. 20–25 cm
 Distal/Proximal of Petal/Sepal
 Aramaki 38°15'30.8"N
 140°50'34.4"E 
      
   Kagitoriyama
 38°14'27.2"N 140°49'30.6"E
 Ranunculaceae
 Aconitum japonicum Thunb. subsp. subcuneatum (Nakai)
 Kadota 3.5–4.5 cm
 Distal/Proximal of Petal/Sepal†
 Tsuta 40°35'52.4"N
 140°57'16.4"E 
      
   Tashiro
 40°41'59.5"N 140°55'10.9"E
 Ranunculaceae
 Aquilegia buergeriana Siebold et Zucc. var. oxysepala (Trautv.
 et C.A.Mey.) Kitam. (purple sepal) 3–3.5
 cm Distal/Proximal of Petal/Sepal
 Hayasaka 39°50'58.2"N
 141°30'29.9"E 
      
   Sodeyama
 40°01'42.3"N 141°32'28.8"E
    
     Hakkoda
 40°38'54.5"N 140°51'07.8"E
   (yellow
 sepal)    
 Hayasaka 39°50'58.2"N
 141°30'29.9"E 
      
   Sodeyama
 40°01'42.3"N 141°32'28.8"E
 Saururaceae
 Houttuynia cordata Thunb. 3–4 cm
 Distal/Proximal of bracts
 Aobayama 38°15'33.0"N
 140°51'20.9"E 
      
   Aramaki
 38°15'31.6"N 140°50'31.2"E
    
    
 Kawauchi 38°15'27.6"N
 140°50'49.1"E 
 † Because petals of Aconitum japonicum are too
 small for separate measurements of chemical traits for distal and proximal
 parts, chemical traits of whole part of petals were measured for the
 species. <strong>Table 2</strong>: List of
 sample segmentation methods for each species. Assigned categories (Sepal
 or Petal and Distal or Proximal) for the floral parts are also
 shown. 
 Family Species
 Segmented part Position
 Part Remarks 
 Asparagaceae
 Hosta sieboldii Tube - Proximal
 Proximal NA Because H.
 sieboldii has gamopetalous flowers, we couldn't separate petals and
 sepals for this species. 
     Tube -
 Middle Distal NA
    
   Tube - Distal
 Distal NA  
 Asphodelaceae
 Hemerocallis fulva Inner petal -
 Proximal Proximal
 Petal   
     Inner petal -
 Distal Distal Petal
    
   Outer petal - Proximal
 Proximal Petal
    
   Outer petal - Distal
 Distal Petal
    
   Sepal - Proximal
 Proximal Sepal
    
   Sepal - Distal
 Distal Sepal
   
 Asteraceae Aster ageratoides
 Petal - Proximal Proximal
 Petal   
     Petal -
 Distal Distal Petal
    
 Aster microcephalus Petal -
 Proximal Proximal
 Petal   
     Petal -
 Distal Distal Petal
    
 Helianthus tuberosus Petal -
 Proximal Proximal
 Petal   
     Petal -
 Distal Distal Petal
   
 Liliaceae Cardiocrinum cordatum
 Lower petal - Proximal Proximal
 Petal   
     Lower petal -
 Middle Distal Petal
    
   Lower petal - Distal
 Distal Petal
    
   Upper petal - Proximal
 Proximal Petal
    
   Upper petal - Middle
 Distal Petal
    
   Upper petal - Distal
 Distal Petal
    
   Upper sepal - Proximal
 Proximal Sepal
    
   Upper sepal - Middle
 Distal Sepal
    
   Upper sepal - Distal
 Distal Sepal
    
   Lower sepal - Proximal
 Proximal Sepal
    
   Lower sepal - Middle
 Distal Sepal
    
   Lower sepal - Distal
 Distal Sepal
    
 Lilium auratum Petal - Proximal
 Proximal Petal
    
   Petal - Distal
 Distal Petal
    
   Sepal - Proximal
 Proximal Sepal
    
   Sepal - Distal
 Distal Sepal
   
 Ranunculaceae Aconitum japonicum
 Lower sepal NA
 Sepal For sample groups having very small
 amounts of distal and proximal parts of lower sepals, samples of proximal
 and distal parts were mixed and the chemical traits were measured. Samples
 in these groups were excluded from the analyses which included position
 terms.  
   Lower sepal - Proximal
 Proximal Sepal
    
   Lower sepal - Distal
 Distal Sepal
    
   Lateral sepal - Proximal
 Proximal Sepal
    
   Lateral sepal - Distal
 Distal Sepal
    
   Spur and petal
 NA Petal Because the
 petals and the spurs were very small, the chemical traits were measured
 using samples consisting of combined distal and proximal parts.
    
 Helmet - Proximal Proximal
 Sepal   
     Helmet -
 Distal Distal Sepal
    
 Aquilegia buergeriana (purple) Petal -
 Proximal Proximal
 Petal   
     Petal -
 Distal Distal Petal
    
   Sepal - Proximal
 Proximal Sepal
    
   Sepal - Distal
 Distal Sepal
    
   Spur
 Proximal Sepal
    
 Aquilegia buergeriana (white) Petal -
 Proximal Proximal
 Petal   
   Petal - Distal
 Distal Petal
     
   Sepal - Proximal
 Proximal Sepal
     
   Sepal - Distal
 Distal Sepal
     
   Spur
 Proximal Sepal
     
 Saururaceae Houttuynia cordata
 Leaf NA NA
 Not used for any analyses. 
     Bract -
 Distal Distal NA
    
   Bract - Proximal
 Proximal NA
  
description: Please see the readme.md or readme.html files in the dataset for
 the details of the dataset.