Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory

Marcel Schrijvers-Gonlag; Christina Skarpe; Riitta Julkunen-Tiitto; Antonio B S Poléo

doi:10.1371/journal.pone.0298229

. 2024 Mar 4;19(3):e0298229. doi: 10.1371/journal.pone.0298229

Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory

Marcel Schrijvers-Gonlag ^1,^*, Christina Skarpe ¹, Riitta Julkunen-Tiitto ², Antonio B S Poléo ¹

Editor: Branislav T Šiler³

PMCID: PMC10911626 PMID: 38437193

Abstract

Herbivory can be reduced by the production of defense compounds (secondary metabolites), but generally defenses are costly, and growth is prioritized over defense. While defense compounds may deter herbivory, nutrients may promote it. In a field study in boreal forest in Norway, we investigated how simulated herbivory affected concentrations of phenolics (generally a defense) and the carbon/nitrogen (C/N) ratio in annual shoots of bilberry (Vaccinium myrtillus), a deciduous clonal dwarf shrub whose vegetative and generative parts provide forage for many boreal forest animals. We measured concentrations of total tannins, individual phenolics, nitrogen and carbon following several types and intensities of herbivory. We identified 22 phenolics: 15 flavonoids, 1 hydroquinone and 6 phenolic acids. After high levels of herbivory, the total tannin concentration and the concentration of these 22 phenolics together (called total phenolic concentration) were significantly lower in bilberry annual shoots than in the control (natural herbivory at low to intermediate levels). Low-intensive herbivory, including severe defoliation, gave no significantly different total tannin or total phenolic concentration compared with the control. Many individual phenolics followed this pattern, while phenolic acids (deterring insect herbivory) showed little response to the treatments: their concentrations were maintained after both low-intensive and severe herbivory. Contrary to our predictions, we found no significant difference in C/N ratio between treatments. Neither the Carbon:Nutrient Balance hypothesis nor the Optimal Defense hypotheses, theories predicting plant resource allocation to secondary compounds, can be used to predict changes in phenolic concentrations (including total tannin concentration) in bilberry annual shoots after herbivory: in this situation, carbon is primarily used for other functions (e.g., maintenance, growth, reproduction) than defense.

Introduction

Bilberry (Vaccinium myrtillus L.) is an abundant species in boreal forests in Fennoscandia [1–3], and a key understory component influencing soil properties and forest regeneration and succession [4]. This deciduous clonal dwarf shrub has evergreen shoots and grows on nutrient-poor soil by ramets, i.e., orthotropic shoots branching from buds on a rhizome [1, 2, 5–8]. In this study, we called the aboveground orthotropic shoot (the stem) including side shoots and leaves a ramet.

Vegetative and generative parts of bilberry are important forage for many mammal, bird, and insect species in all seasons [9–16]. One of many strategies to minimize herbivory on vegetative plant tissue is the production of defense compounds (defense metabolites, secondary compounds) by plants [17, 18]. Under resource limitation, trade-offs occur among growth, maintenance, storage, reproduction, and defense in plants [19, and references herein]. Some of the theories that predict plant resource allocation to secondary compounds are relevant for bilberry-herbivore interactions. In this study we focused on the Optimal Defense hypotheses (consisting of several (sub)hypotheses, see [20]) and the Carbon:Nutrient Balance hypothesis.

The Optimal Defense hypotheses predict that production of inducible defenses is low when herbivory is absent or nearly absent and increases when herbivory is present, as defenses are costly [20–28]. In general, however, growth is prioritized over defense [19, 29–32, but see 33, and references herein]. Therefore, a severe loss of photosynthetic tissue may not allow for production of defense compounds and may even lead to the breakdown of existing defenses, resulting in lower resistance to herbivory [34–37].

Like many deciduous woody species growing on nutrient-poor soils, bilberry stores carbon in roots and other woody tissue, like stems [19, 30, 38, 39]. The Carbon:Nutrient Balance (CNB) hypothesis predicts a mobilization of these carbon reserves and an increase in the level of carbon-based defense compounds in bilberry after herbivory on shoots and leaves [19, 20, 38, 40]. Phenolic compounds (phenolics) are primarily composed of carbon [41] and can act as such carbon-based defense compounds reducing herbivore performance and herbivory [42–47]. Phenolics include tannins (condensed tannins or proanthocyanidins and hydrolyzable tannins), flavonoids and other small molecular mass phenolics, including cinnamic acids [41, 42, 48]. Many different phenolics have been identified in bilberry stems, shoots, leaves, berries, and rhizomes [49–55]. We expected that the effects of tissue damage, resulting from herbivory or other causes, on phenolic concentration in bilberry vary depending on several factors: the damage type (whether leaves, shoots or the whole ramet is damaged), damage intensity, and the level the actual phenolic can function as a defense against herbivores, as different phenolics have multiple biological functions and efficacy [56–63].

Defense compounds may deter herbivory, while nutrients may promote it [18, 64–68]. Nitrogen concentration in bilberry, which is often used as a proxy for nutrient concentration, increases after browsing in several woody species, often regardless of soil productivity [69–73]. Pruning, the partial or complete removal of stem and/or shoots, reduces bud numbers and increases the root:shoot ratio. This results in decreased competition for nutrients among meristems, causing an increase in new plant tissue nutrient concentration [32, 74–78]. On the other hand, severe defoliation results in a loss of nitrogen [79], or at least in the loss of proportionally more nutrients than carbon, as most nutrients are found within the foliage of deciduous species in the growing season [19]. Furthermore, severe defoliation results in increased fine root mortality [80, 81]. This leads to reduced nutrient absorption which results in a decreased nutrient concentration, especially on nutrient-poor soils [82, 83]. Therefore, we expected that the effects of tissue damage, due to herbivory or other causes, on nitrogen concentration in bilberry vary, depending on type and intensity of damage.

Most research on phenolics in bilberry has focused on berries, although studies on leaves, shoots and stems have been conducted [49, 50, 54, 84–86]. Previous studies of herbivory, nutritional quality and defense responses of bilberry shoots and leaves did not involve controlled clipping experiments, nor measurements of phenolic, nitrogen and carbon concentrations in annual shoots [87–92]. After herbivory, we expected a measurable response in the young tissue of annual shoots [21, 39, 93]. For these reasons, we investigated how simulated herbivory affected phenolic concentrations and the carbon/nitrogen (C/N) ratio, often used as indicator of plant nutritional quality [94, 95], in bilberry annual shoots. We measured total tannin concentration and concentrations of individual phenolics, nitrogen and carbon in bilberry annual shoots after several types and intensities of simulated herbivory. Persson and colleagues [55] performed a simulated browsing study on bilberry investigating responses in phenolic and nitrogen concentrations and C/N ratio in leaves and leafless shoots. Different from Persson and colleagues, who performed different levels of simulated moose (Alces alces L.) browsing only, we used three types of simulated herbivory, representing herbivory by large ungulates (eating ramets), herbivory by smaller mammals, birds and insects (eating annual shoots) and herbivory by insects (eating leaves). Our study was performed under ambient herbivory conditions in boreal forest in southeastern Norway in 2014.

Our objective was to examine how different herbivory types (ramet herbivory, annual shoot herbivory, leaf herbivory) and intensities affect the concentration of phenolics (total tannins as well as several small molecular mass phenolics) and nitrogen (nutritional quality) in bilberry annual shoots. We compared our simulated herbivory (from here often just called herbivory) with ambient herbivory, which was at a low to intermediate level. We considered our results in the context of the plant defense theories described above. We predicted that in bilberry annual shoots, the concentration of:

I. phenolics is, at low to intermediate herbivory levels, positively correlated with intensity of herbivory;
II. phenolics is, at high herbivory levels, lower than without herbivory;
III. nitrogen is, at low to intermediate herbivory levels, positively correlated with intensity of herbivory, i.e., the C/N ratio is negatively correlated with intensity of herbivory;
IV. nitrogen is, at high herbivory levels, lower, i.e., the C/N ratio is higher, than without herbivory.

Methods

Study area

We conducted our study in coniferous boreal forest at six locations (400–670 m a.s.l.) in the Østerdalen valley close to Evenstad (latitude 61.43 °N, longitude 11.08 °E) in southeastern Norway in 2014. In this year, mean annual temperature was 4.8 °C and total precipitation was 896 mm [96]. The forest was owned by the Norwegian state-owned land and forest enterprise Statskog SF (www.statskog.no), who granted permission to do the experiment, including sampling bilberry plants.

Study design

Field treatments

At each location, we used four lines, more or less parallel and spaced by ten m, to select bilberry ramets with approximately two m distance between consecutive ramets (Fig 1). Along each line, we selected 33 or 34 ramets at the beginning of the growing season (May) and marked them with steel wire. Selected ramets had at least ten shoots longer than 1.0 cm from the previous growing season (annual shoots from 2013, S1 File), and no visual signs of extensive herbivory (most ramets had some past herbivory signs), so the initial herbivory level for all ramets was low. In total we selected 135 ramets at each location. We divided the ramets within each location randomly (S1 File) into four treatment groups: control (n = 30), abbreviated to C, representing ambient, initially low, herbivory only; ’leaves cut’ (n = 45), abbreviated to L, representing additional herbivory by insects; ’annual shoots cut’ (n = 45), abbreviated to S, representing additional herbivory by insects and small-sized vertebrates; and ’ramet cut’ (n = 15), abbreviated to R, representing additional herbivory by large ungulates. At all six locations, we removed leaves by hand (treatment L) at three different intensities: 10% from 15 ramets, 50% from another 15 ramets and 100% from the remaining 15 ramets (S1 File). At five locations, we removed annual shoots by hand (treatment S) at similar intensities (10%, 50% and 100%; n = 15 for each), and we cut the ramet in treatment R by removing 90% of the ramet with garden scissors (Fig 1). In total this resulted in eight treatments: ’control’ (C: ambient, initially low, herbivory), ’leaves cut’ (3 intensities: L10, L50, L100), ’annual shoots cut’ (3 intensities: S10, S50, S100), ’ramet cut’ (R). Ramets in C that experienced severe herbivory between selecting and harvesting, were excluded from our analyses: therefore, all control ramets experienced herbivory at low to intermediate levels (ambient herbivory). The shoots were removed and ramets cut on 24–27 May and leaves were removed in the period 21 June– 2 July. In our experiment, we considered C as herbivory at the lowest level. Within L, L10 represented leaf herbivory at a low level, L50 represented leaf herbivory at an intermediate level and L100 represented leaf herbivory at a high level. Similar with S: within S, S10 represented annual shoot herbivory at a low level, S50 represented annual shoot herbivory at an intermediate level and S100 represented annual shoot herbivory at a high level. We considered R as herbivory at the highest level and S100 as herbivory at the second highest level in our experiment: judging after proportion of biomass removed, these two treatments were the two most severe herbivory treatments in our study.

Fig 1 — Schematic bilberry ramet, eight treatments (see text): C (control), three leaf treatments (L), three shoot treatments (S), one ramet treatment (R). 21 annual shoots are visible (1 at the top of the stem, 10 at each side of the stem). AGE: the stem and all shoots (same schematic bilberry as in the treatments) are depicted with numbers: the stem is at least four years old and indicated with 4, shoots at least three years old are indicated with 3, shoots at least two years old are indicated with 2, annual shoots (with leaves) are indicated with 1. NOTE: the upper three-year-old shoot at the right side of the stem and the middle three-year-old shoot at the left side of the stem may also be two years old. LOCATION: at four lines, 34 (upper three lines) and 33 (lower line) ramets are selected: 30 control ramets and 15 ramets for every one of the other 7 treatments, randomly appointed. Approximately 2 m between each ramet and 10 m between each line. This location is one of six locations (namely: “Imsdalen 1”). Drawing: Marcel Schrijvers-Gonlag.

Bilberry sampling

All ramets (n = 750) were harvested towards the end of the growing season (19–28 August) by cutting the stem at ground level with garden scissors. The ramets were dried for minimum 48 hours in a drying oven (Binder FED 720 E2, Germany) at 30 °C, before the dried ramets were stored in a dark and dry place at room temperature. From each location, we randomly selected a minimum of five dried ramets from each herbivory treatment (including control), resulting in 232 ramets in total. From each of these ramets, we randomly selected five annual shoots (S1 File), continued drying these annual shoots for minimum 24 hours at 30 °C and stored them in a dark and dry place at room temperature, prior to preparation and analyses of tannin, phenolic, carbon and nitrogen concentrations.

Chemical analyses

Bilberry shoots

Before analyses of acetone-soluble tannins, methanol-soluble phenolics, and total carbon and nitrogen, the shoots were cut in fragments of maximum 0.5 cm and for each ramet we transferred these subsamples to a 2 ml or 7 ml vial with three stainless steel beads (2.8 mm) to pulverize the tissue; with large subsamples (approximately 200, 300 and 400 mg; all weight measurements in this study: scale Sartorius SE2, d = 0.1 μg) we used four, five or six beads, respectively. The shoot fragments were pulverized by the beads using a Precellys 24 homogenizer (Bertin Technologies, France): 25 s at 5500 rpm, 15 times with two minutes in between. When handling the shoots, we used disposable latex gloves.

Shoot tannins

The shoots were analyzed for acetone (70%)-soluble tannins (e.g., hydrolyzable tannins and polymeric condensed tannins (proanthocyanidins)) [97, 98: S1, 99] with a spectrophotometer (Spectronic 20 Genesys; Spectronic Instruments, USA). We slightly adjusted the acid butanol assay for proanthocyanidins [100] to measure tannins in our subsamples (S2 File). To relate tannin concentration in our subsamples to measured absorbance (at 550 nm) we built a standard reference curve, using Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) for tannin purification [98: S1, 101].

Shoot phenolics

Methanol-soluble phenolics (e.g., flavonoids and phenolic acids) were extracted from the shoots and quantified using high performance liquid chromatography (HPLC) with injection volume 10 μl (Agilent series 1100) and identified using a UHPLC quadrupole time-of-flight liquid chromatograph–mass spectrometer (Agilent Technologies, 6540 UHD Accurate-Mass Q-TOF LC/MS, 1290 Infinity) as described by Nissinen and colleagues [102] (S3 File). Compounds that could not be identified were not used in this study. We used D(-)-Salicin min. 99% CHR (Aldrich-Chemie, West-Germany) in methanol (100%) as an internal standard in two out of five subsamples to evaluate extraction efficiency (S3 File).

Shoot carbon and nitrogen

The shoots were analyzed for carbon and nitrogen (total concentration (mg/g, dry weight) after destruction; micro CN-analyzer (thermo), Chemical Biological Soil Laboratory (quality system based on the ISO-17025 standard), Wageningen University, July 2016).

Statistical analyses

The total tannin absorbance measurements were averaged per subsample and with the standard reference curve and subsample weight these subsample means were converted to concentrations (mg tannins/g shoots, dry weight), which were used in further analyses. In our HPLC analyses we used the concentration (mg/g, dry weight) of every identified phenolic as the response variable in our modeling, calculated as: ((rf x area) / weight) / (inj / tot) where rf is the HPLC response factor for the actual phenolic at the used wavelength, area is the peak area in the HPLC result table (mAU*s) at the used wavelength, weight is the initial shoot subsample weight (mg), inj is the HPLC injection volume (10 or 15 μl) and tot is the total volume (600 μl) in which the subsample was dissolved (300 μl methanol + 300 μl purified water, S3 File). The HPLC response factor is the ratio between the concentration of a specific compound (mg/g) and the response of the detector (area: mAU*s) to this compound at a specific wavelength; we used response factors previously determined using standards with known concentrations (S1 Table). Before analyses, phenolic concentrations were converted to 100% to recover losses in the extraction procedure (S3 File). When no value in the HPLC result table was present for a phenolic, we used a concentration of 0 mg/g, although often a small peak on the HPLC chromatogram was visible.

Differences between treatments were investigated with a one-way ANOVA test. In all ANOVA analyses we used equal sample sizes across groups, to avoid inflation of error rates and to guarantee homogeneity of variance [103]. If necessary, samples were randomly removed to obtain balanced sample sizes. We used the total tannin concentration, the concentration of each identified phenolic and the concentration of all identified phenolics together as response variables in predictions I and II. We also used a one-way ANOVA test to investigate differences between treatments on the response variable C/N ratio (predictions III and IV). When the ANOVA test indicated a significant difference (we used a significance level of 5%), differences between groups were investigated with Tukey’s HSD post-hoc test. We used the package ‘emmeans’ in the software ‘R’ to calculate some general statistics and to further investigate the relationship between several response variables and treatments [104, 105]. Figs 2–5 were created with the R-package ‘svglite’ [106] and the software ‘Inkscape’ (version 1.2.1). All model analyses were performed in R (version 4.1.2, 4.2.2, 4.2.3 and 4.3.1) [105].

Fig 2 — Boxplot with total concentration (mg/g, dry weight), n = 200, every treatment n = 25. Treatments: see text. The bottom and top of each box indicate the first and third quartiles. Bold horizontal lines within each box indicate median values. The plot whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range away from the box; extreme data points more than 1.5 times the interquartile range away from the box are indicated with black points. Treatments with the same letter above the box are not different from each other (P > 0.05).

Fig 5 — Boxplots with individual phenolic concentration (mg/g, dry weight). Only phenolics with at least one treatment significantly different from another treatment are shown (n = 200, every treatment n = 25). Treatments: see text. The bottom and top of each box indicate the first and third quartiles. Bold horizontal lines within each box indicate median values. The plot whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range away from the box; extreme data points more than 1.5 times the interquartile range away from the box are indicated with black points. Treatments with the same letter above the box are not different from each other (P > 0.05).

Results

Shoot tannins

On average, bilberry annual shoots consisted of 25–30% tannins (dry weight; Table 1). The intensity of herbivory affected tannin concentrations (ANOVA: F_7,192 = 6.18, P < 0.001; Fig 2; Table 1). S100 and R resulted in significantly lower tannin concentrations than C. All other treatments did not differ significantly from each other nor from C (Fig 2).

Table 1. Tannins, phenolics, carbon, nitrogen, and C/N in bilberry annual shoots after simulated herbivory.

Mean concentration (mg/g ± se (standard error of the sample mean), dry weight) of tannins, 22 phenolics (see text), carbon (C) and nitrogen (N) and mean C/N ratio (± standard error), per treatment (see text). For all treatments together (All) also the sd (standard deviation of the sample) is given. Number of observations between parentheses.

Treatment	Tannins	Phenolics	Carbon (C)	Nitrogen (N)	C/N
C	291.1 ± 7.1 (33)	106.4 ± 4.1 (33)	498 ± 2.0 (33)	12.27 ± 0.36 (33)	41.9 ± 1.2 (33)
L10	278.2 ± 7.1 (33)	106.1 ± 4.2 (32)	497 ± 2.0 (33)	12.40 ± 0.36 (33)	40.9 ± 1.2 (33)
L50	275.7 ± 7.2 (32)	100 9 ± 4.2 (32)	498 ± 2.1 (32)	12.64 ± 0.37 (32)	40.1 ± 1.2 (32)
L100	274.4 ± 7.2 (32)	101.4 ± 4.2 (32)	498 ± 2.0 (32)	12.26 ± 0.37 (32)	41.7 ± 1.2 (32)
S10	292.8 ± 8.2 (25)	110.2 ± 4.7 (25)	500 ± 2.4 (25)	12.17 ± 0.42 (25)	42.1 ± 1.4 (25)
S50	257.1 ± 8.2 (25)	88.6 ± 4.7 (25)	498 ± 2.4 (25)	12.94 ± 0.42 (25)	39.6 ± 1.4 (25)
S100	249.3 ± 8.0 (26)	84.9 ± 4.6 (26)	504 ± 2.3 (26)	12.76 ± 0.41 (26)	40.7 ± 1.4 (26)
R	235.7 ± 8.0 (26)	75.1 ± 4.6 (26)	500 ± 2.3 (26)	12.62 ± 0.41 (26)	41.3 ± 1.4 (26)
All	270.5 ± 2.9, sd = 44.1 (232)	97.5 ± 1.7, sd = 25.9 (231)	499 ± 0.8, sd = 11.8 (232)	12.50 ± 0.14, sd = 2.08 (232)	41.0 ± 0.5, sd = 6.9 (232)

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Shoot phenolics

We identified 22 phenolics: 15 flavonoids, 1 hydroquinone and 6 phenolic acids (Fig 3). The recovery of the internal standard was around 95%: min = 58.9%, mean = 94.5%, max = 107.5%, sd = 6.1% (n = 90). In one subsample the recovery of the internal standard was 58.9%, quite different from all others. Therefore, we excluded this subsample from the phenolic analyses. Without this subsample, the recovery of the internal standard changed to: min = 83.1%, mean = 94.9%, max = 107.5%, sd = 4.7% (n = 89).

The phenolic concentration of all these 22 identified phenolics together (analyzed together) is from here called total phenolic concentration. On average, almost 10% of bilberry annual shoots consisted of these 22 phenolics (dry weight; Table 1). Compared to C and L10, which had very similar total phenolic concentrations, all other treatments except S10 resulted in lower mean total phenolic concentrations (Table 1; S2 Table). The differences between C, L10 and S10 were not significant (Fig 4), but a significant difference in total phenolic concentration between one or more other treatments was present (ANOVA: F_7,192 = 6.64, P < 0.001). Within L, the total phenolic concentration did not differ significantly, but it did within S (Fig 4). S100 and R resulted in significantly lower total phenolic concentrations than C. R resulted in the lowest mean (S2 Table) and median (Fig 4) total phenolic concentration.

Fig 4 — Boxplot with total phenolic concentration (mg/g, dry weight), n = 200, every treatment n = 25. Treatments: see text. The bottom and top of each box indicate the first and third quartiles. Bold horizontal lines within each box indicate median values. The plot whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range away from the box; extreme data points more than 1.5 times the interquartile range away from the box are indicated with black points. Treatments with the same letter above the box are not different from each other (P > 0.05).

As the total phenolic concentration is the sum of all identified phenolic concentrations, many of these individual phenolics showed a similar pattern: R resulted in the lowest mean phenolic concentration in 15 phenolics (68%). Considering R and S100 together, this number increased to 19 phenolics (86%) (S2 Table). Investigating significant differences between treatments, one or more herbivory treatments resulted in significantly different phenolic concentrations in nine phenolics; phenolic acids showed little response to the treatments (Table 2). Considering the leaf (L) removal treatments only, there were no significant differences in individual phenolic concentration between treatments, nor were any L treatments significantly different from C (Fig 5). Considering the annual shoot (S) removal treatments, in two phenolics (procyanidin 2 and 4) the phenolic concentration was significantly lower in S100 than in S10. Compared to the other treatments, S10 resulted in the highest mean phenolic concentration in eight phenolics (36%) and in the total phenolic concentration (S2 Table), although no significant difference between S10 and C was present in any of the phenolics. In three phenolics (epicatechin, procyanidin 2 and quercetin 3-glucuronide) S100 resulted in a significantly lower concentration than C (Fig 5). In seven phenolics, R resulted in a significantly lower phenolic concentration than C (Fig 5).

Table 2. Differences in individual phenolic concentration in bilberry annual shoots between simulated herbivory treatments.

The number of differences (#) between treatments (P < 0.05) is given for 22 phenolics separately, and for all 22 phenolics analyzed together (n = 200, every treatment n = 25).

Flavonoids	#
Epicatechin	6
Gallocatechin derivative	ns
Hyperin	0
Isorhamnetin 3-glucoside	ns
Kaempferol 3-glucoside	ns
Monocoumaroyl-isoquercitrin¹	ns
Procyanidin 1	1
Procyanidin 2	8
Procyanidin 3	ns
Procyanidin 4	6
Procyanidin 5	4
Procyanidin 6	ns
Quercetin 3-arabinoside	2
Quercetin 3-glucuronide	5
Quercitrin	ns
*Sum flavonoids*	32
Hydroquinones
Arbutin derivative	7
*Sum hydroquinones*	7
Phenolic acids
Chlorogenic acid	ns
Cinnamic acid derivative	ns
Para-hydroxycinnamic acid derivative 1	ns
Para-hydroxycinnamic acid derivative 2	ns
Para-hydroxycinnamic acid derivative 3	ns
Protocatechuic acid derivative	1
*Sum phenolic acids*	1
*Sum all individual phenolics*	40
All 22 phenolics together	9

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¹Monocoumaroyl-isoquercitrin: identification uncertain.

If the ANOVA test result P < 0.05 but the Tukey’s HSD test gave only P_adj values > 0.05 a ‘0’ is shown in the table. ANOVA test results P > 0.05 show ‘ns’ in the table (irrespective of the Tukey’s HSD test result).

Shoot carbon and nitrogen

On average, half of the bilberry annual shoots (dry weight) consisted of carbon and slightly over 1% consisted of nitrogen (Table 1). There was little variation in carbon and nitrogen concentration and in C/N ratio between treatments (Table 1). We found no significant difference in C/N ratio between treatments (ANOVA: F_7,192 = 0.40, P = 0.90). Analyses of carbon concentration and nitrogen concentration yielded similar results: no significant difference between treatments (carbon: ANOVA: F_7,192 = 1.04, P = 0.40; nitrogen: ANOVA: F_7,192 = 0.37, P = 0.92).

Discussion

Shoot tannins and total phenolics

The two most severe herbivory treatments, concerning removed biomass, resulted in significantly lower tannin and total phenolic concentrations than the control, while less severe herbivory treatments did not differ significantly from the control. These results support our second prediction but suggest that little to intermediate loss of photosynthetic tissue does not increase carbon-based defense compound concentrations in bilberry, contrary to our first prediction. Several factors may contribute to these results.

First, not all phenolics respond to herbivory as predicted by the CNB hypothesis, as many phenolics compete with proteins for the nitrogen containing precursor phenylalanine as described by Jones & Hartley [107] in their Protein Competition Model (PCM) [42, 108, 109]. As the boreal forest is a nitrogen-limited ecosystem [110–113], competition for nitrogen between biosynthesis of proteins and of many phenolics is expected in boreal forest ecosystems. Therefore, the nutrient-poor soil may not provide sufficient nitrogen for bilberry to increase these secondary compound concentrations while continuing protein demanding primary processes as growth and reproduction.

As bilberry is a clonal plant, connected ramets may translocate compounds from nondefoliated to defoliated ramets, as has been documented in perennial graminoid species and herbs [114–116, and references herein]. Translocation of carbohydrates or even phenolics between connected ramets may be another reason for the absence of a correlation between leaf herbivory and subsequent phenolic concentrations in bilberry annual shoots. Also, high fine root mortality may not, or to a lesser extent, occur in clonal bilberry, which possibly translocates carbohydrates between connected ramets to maintain its root activity.

Furthermore, our simulated leaf herbivory (mechanical wounding by hand) is not equivalent to leaf herbivory by insects, birds, or small mammals [27, 117–125, reviewed by 126]. Although most of these studies indicate that simulated leaf and shoot herbivory performed by mechanical wounding induces a less pronounced plant response, such simulated herbivory generally does induce a plant response [see the aformentioned references and 87, 127, 128], as the general response of plants to wounding and herbivore damage is essentially the same [129]. This is particularly true in nutrient-poor sites [130], like our study system. Nevertheless, this indicates that bilberry responses to simulated leaf herbivory may differ from responses to natural herbivory, which can further contribute to our observed absence of a correlation between simulated leaf herbivory and subsequent phenolic concentrations in bilberry annual shoots.

Additionally, this observed absence can be due to other reasons. A response can have been counteracted by transport of existing phenolics from shoots to leaves, as some plant species store phenolics in shoots which are transported to leaves following herbivory [127]–although such reallocation of phenolics may not be very important [107]. Furthermore, the time between our leaf herbivory treatments and bilberry ramet harvesting was 48–68 days. Possibly, bilberry only responds with a short-term response that was no longer detectable after 48 days. For instance, in another woody species, the condensed tannin concentration returned to pre-herbivory values less than 66 hours after herbivory [131, see also 132]. An alternative possible reason is a very delayed response: responses remain undetectable until at least 68 days after the treatment. This last option seems very unlikely in terms of plant fitness, but cannot be ruled out with the data available. Experiments measuring how long induction lasts in different bilberry tissues are needed to support or reject these speculations.

Another possible reason for our observed results is that an herbivory-induced change in phenolics occurs in other plant parts, e.g., leaves, and is not detectable in annual shoots. This seems unlikely, as Persson and colleagues found that bilberry leaves and bilberry leafless shoots were comparable in their response to simulated moose herbivory, at least for flavonoids and condensed tannins [55]. In contrast with our results, Persson and colleagues found an increase in flavonoid and condensed tannin concentration in bilberry shoots with increasing simulated moose herbivory. Possibly their results were influenced by a side-effect of the treatment: a more open canopy resulted in more solar radiation which could have induced production of secondary compounds, as has been found and discussed in other studies [50, 55, 133–139, and references herein].

Shoot individual phenolics

In seven phenolics, R resulted in a significantly lower phenolic concentration than C, while in fifteen phenolics no significant difference between the control and other treatments was present. The concentrations of all phenolic acids found in our study were unaffected by severe herbivory. Interestingly, these phenolics are known to deter herbivory by insects: all reduce larval growth rate, some also promote larval mortality and chlorogenic acid even shows strong anti-nutritive properties against various invertebrate herbivores, including adult beetles and grasshopper nymphs [45, 140–147]. This indicates that bilberry responds to severe herbivory by maintaining concentrations of phenolics which deter herbivory on a certain level. As we could not find information about biological functions related to herbivory for other specific phenolics identified in our study, we don’t know how bilberry responds to severe herbivory in the case of phenolics which promote herbivory (possibly by decreasing their concentrations?). Herbivory experiments with specific phenolics are necessary to support or reject these speculations.

We did not find (+)-catechin in our bilberry annual shoots, as has been found in other bilberry studies [49, 50]. In the HPLC chromatogram (Fig 3), (+)-catechin, if present, comes shortly after chlorogenic acid. This means that when a large quantity of chlorogenic acid is present, as with our subsamples (Fig 3, S2 Table), the chlorogenic acid peak overlaps with the peak of (+)-catechin and it is not possible to separate the latter from the former, especially when only little (+)-catechin is present. Therefore, unidentified amounts of (+)-catechin may have been present in our subsamples, but if so, (+)-catechin was present in much lower amounts than epicatechin (S2 Table).

Shoot carbon and nitrogen

The carbon and nitrogen concentrations and C/N ratio in our study are comparable with results from other studies [6, 50, 136, 148, 149] but differ from bilberry nitrogen concentrations found by Selås and colleagues [150]. Our results show that both the carbon and nitrogen concentration, as well as the C/N ratio, in bilberry annual shoots are not affected by herbivory. These findings do not support our predictions III and IV. Apparently, mechanisms that either increase or decrease nutrient concentration after herbivory (see Introduction), cause this overall result. Additionally, in clonal bilberry carbohydrates may be translocated from source ramets to connecting ramets under herbivory pressure, and to their root system, to compensate for a lack of carbon (see before). This may prevent an increase in fine root mortality and, consequently, a decrease in nutrient concentration. Thus, clonality can further explain the lack of support for our predictions III and IV.

Another possible reason is, as with phenolics (see before), that a change in C/N ratio does not occur in bilberry annual shoots but in other plant parts, e.g., leaves, as shown in other woody species [70, 72, 75, 95, 151] (although Laine and Henttonen [148] did not find a correlation between microtine density and nitrogen concentration in bilberry leaves). As we do not have data about carbon and nitrogen concentrations in plant parts other than annual shoots, we cannot rule out this possibility.

Finally, Flower-Ellis [6] reported much variation in nitrogen concentration between long, vegetative shoots and short, predominantly flowering shoots, as well as in ramets from different ages and positions in the stand (causing variation in light and water conditions). Such variation may obscure effects from herbivory.

Study design

In this study, we removed annual shoots in four treatments, at different intensities: S10, S50, S100 and R. Only with the last two treatments (S100 and R), all (or almost all in some R treatments) annual shoots were removed. Approximately three months after removal, we harvested annual shoots from the ramets: therefore, only with S100 and R the harvested annual shoots were all (or almost all in some R treatments) new shoots, grown after the clipping event had occurred. In all other treatments, most likely the analyzed annual shoots had all (L treatments) or partly (S10: around 90 percent, S50: around 50 percent) been present at the ramet before the clipping event took place. Interestingly, only at high herbivory levels (S100 and R), we found a significant difference in tannin concentration and total phenolic concentration compared to the control. This means that all treatments from which we analyzed many older annual shoots (from before the clipping event) did not yield a significant difference in phenolic concentration in the annual shoots compared to the control. Although many of these annual shoots probably were not fully grown at the time of clipping and therefore also their tissue had (partly) developed after the clipping event took place, this means that we analyzed annual shoots in S10, S50 and all L treatments, that were present before the clipping event occurred–at least an important part of them. If a chemical response to the treatment does not occur in older shoots but only, or mainly, occurs in new tissue (this we don’t know) this shortcoming in our study design has serious consequences for our results regarding to the S10, S50 and all L treatments.

Defense and other metabolic processes

As our results do not support our first prediction (I: phenolic concentration is, at low to intermediate herbivory levels, positively correlated with intensity of herbivory) but do support our second prediction (II: phenolic concentration is, at high herbivory levels, lower than without herbivory), we conclude that after herbivory, bilberry uses carbon primarily for functions other that defense. This is no more than a speculation, as we have no metric of growth (as total biomass or compensatory growth), or metabolic processes other than phenolic concentrations. Possibly, little herbivory may be almost inconsequential for plant fitness and responses may be absent, or non-detectable, or only morphological, not chemical. Severe herbivory may force bilberry to divert resources from other pools, as existing defense chemical compounds, to compensate for biomass losses. Experiments which specifically focus on morphological responses (as compensatory growth) and reproduction, preferably also chemical responses, after herbivory, are needed to support or reject our speculation.

Conclusions

We conclude that neither the Carbon:Nutrient Balance hypothesis nor the Optimal Defense hypotheses can be used to predict changes in phenolic concentrations (including total tannin concentration) after herbivory in bilberry annual shoots. After herbivory, bilberry uses carbon primarily for functions other than defense (e.g., maintenance, growth, reproduction). Herbivory experiments focusing on morphological responses and reproduction are necessary to further investigate this response. Furthermore, we conclude that bilberry responds to severe herbivory by maintaining concentrations of specific phenolics, which deter herbivory, on a certain level, while decreasing concentrations of other phenolics. Herbivory experiments with specific phenolics, to clearify their function as anti-herbivore compound (i.e., do they affect bilberry’s palatability to herbivores), are necessary to further investigate this response.

Supporting information

S1 File. Annual shoots and random selection.

(PDF)

pone.0298229.s001.pdf^{(89KB, pdf)}

S2 File. Standard reference curve and tannin color test.

(PDF)

pone.0298229.s002.pdf^{(228.9KB, pdf)}

S3 File. Quantifying phenolics using HPLC.

(PDF)

pone.0298229.s003.pdf^{(154.2KB, pdf)}

S1 Table. Response factors.

(PDF)

pone.0298229.s004.pdf^{(100.4KB, pdf)}

S2 Table. Phenolic concentrations after simulated herbivory.

(PDF)

pone.0298229.s005.pdf^{(522.5KB, pdf)}

Acknowledgments

We thank Anne Mehlhoop, Bernardo Toledo González and Marieke Gonlag-Schrijvers for lab work and analytical insights, Sinikka Sorsa, Katri Nissinen, Virpi Virjamo and Md. Nazmul Hasan for help in the lab, Anne Mehlhoop, Andreja Kovše and Umer Qureshi for soil sampling, Maria Greger for initial advice on soil sampling and phenolics, Gé van Steijn for comments on the analyses, and Jo Inge Breisjøberget and Kjell Anders Vikan for information about and digital maps based on the H40 system. We thank the Norwegian state-owned land and forest enterprise Statskog SF for permission to do the experiment on their property. MSG thanks the Stack Exchange Q&A web communities Stack Overflow and Cross Validated for invaluable statistical and analytical insights, Marieke Gonlag-Schrijvers for her patience and understanding, and Ole Arne Hagen / Linnea AS for facilitating a productive working environment during a pandemic.

Data Availability

The datasets and scripts used for the presented analyses are stored in the DataverseNO database and available at https://doi.org/10.18710/U8S3XX.

Funding Statement

This study is a part of the BEcoDyn project, supported by Inland Norway University of Applied Sciences and by a grant from the Norwegian Research Council to Harry P. Andreassen (who sadly passed away in 2019) (NFR project 221056; https://prosjektbanken.forskningsradet.no/en/project/FORISS/221056). MSG received a grant from the Extensus Foundation. Work done by MSG was only partly funded. The Norwegian Research Council (https://www.forskningsradet.no/en/) and the Extensus Foundation (http://www.extensus.nu/index_eng.php) had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0298229.r001

Decision Letter 0

Branislav T Šiler

27 Apr 2023

PONE-D-23-06554Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivoryPLOS ONE

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Reviewer #1: The manuscript entitled Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory cover an interesting topic about the allocation of phenolic compounds during differents scenarios of herbivory. It is in general well written.

However there are some minor aspects that need to be taken to be ready to publish

Why no to use another statistic test such as GLM to test interactions between location and soil productivity

Don you think table 1 should be better in supplementary material?

I think is more informative to add the letters according to a post hoc test in table two. And why are you adding the column of all treatments together? It is useful??

Is it on the guidelines or why the figure legends are in the text?

I don’t think that is necessary to add SE and SD from you data every time you describe the results

I think first paragraph of discussion should be a summary of the results, why start with one particular compound?

When you make your comparisons with other systems, why do not you use related species instead of mountain birch and mopane? there are some interesting articles of other vaccinium species that can be more comparable to your study system

Reviewer #2: I reviewed the paper entitled ‘Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory’. Authors identified several hypotheses related to phenolics in response to simulated herbivory. Principally, related to whether these defenses follow the C:nutrient balance hypothesis or Optimal Defense Theory. The actual experimental data was quite straightforward. Various phenolic compounds and C:N ratios were measured in bilberry shoots. That being said, in my view there is excessive text and lots of speculation that goes far beyond what was tested in this manuscript. The protein competition model is quite interesting, however not properly tested. I am not sure how you can test the protein competition model without measuring any protein…There are many reasons why phenolics would be reduced or unchanged. Did you see differences in protein quantity/quality under simulated herbivory? Further, the conclusion that this may be due to clonality is likewise interesting, but was not tested. Did you look at C:N ratios in clonal ramets connected to experimental ramets, for example, to see whether there may be a flux or allocation of resources to damaged plants. Additionally, why was the foliar content not measured? Especially considering you had a defoliation treatment, one might expect differences to be most apparent in leaves. Authors should justify why this was.

What was actually found, as I understand, is that, other than essentially complete removal of aboveground tissue, your treatments do not reduce specific constitutive phenolics, whereas under severe damage, shoots had lower phenolics, presumably because carbon is being used for regrowth of tissue growth and is limiting. Under ambient herbivory, carbon may not be limiting due to the fact that only minor additional growth is necessary to reach static conditions, and enough is C is thus available for phenolics biosynthesis. Did you have data for changes in biomass? Although the takeaway message is rather simple, I found the MS slightly challenging to read. And was distracted by a lot of possibilities and details that were not tested in the experiments. Perhaps if the paper was reframed and simplified to highlight what was tested, as opposed to what was not (all of these trade-off theories) it would be easier to follow. For example, your first prediction is not even possible to test, because you don’t detect any gallic acid-derived phenolics at all. As such, it doesn’t make much sense to include it in the MS. Your first prediction might instead be something like ‘determine whether bilberry has these compounds at all, and compare them with Phe-derived phenolics’. Then you might be able to contextualize this in the protein competition model. However, because they are not present, this is of course not possible.

I did not leave specific, detailed comments, as lines were not numbered. In subsequent drafts I would recommend adding line numbers.

Reviewer #3: After reading this study several times I can only conclude that to have a chance to be publishable, the manuscript needs to be completely rewritten to address two major problems: 1) a weak initial study design that prevents conclusions to be made with confidence followed by a modification that does little to improve the scientific integrity, and 2) a writing style that is difficult to follow due to a lack of flow and clarity arising from awkwardly constructed sentences and arguments, excessive repetition and inclusion of unnecessary text and information.

With respect strengthening the design of the study, I recommend removing the issue of high- and low productivity sites. The definition of productivity appears to be defined and supported data-wise with one variable, total nitrogen. This information comes from one composite sample per site (n=1). To merge your data and compare this to the one location which “behaved differently” also weakens your study in my opinion. I suggest you remove the idea of site productivity as a factor and rewrite the manuscript based on your “herbivory treatments”; something for which you have much better resolution and data from which to draw more confident conclusions. Present the plant chemistry data (phenols, tannins, N, C, and C/N) together in one Table or Figure, so that the reader can better visualize possible relationships among these variables (for example, see Nosko et al 2020, https://doi.org/10.1016/j.foreco.2019.117839). In your discussion, you can then focus on browsing treatments as they affect the relationships among plant chemistry variables. In the absence of such relationships, some inspiration might be obtained from Warbrick et al 2020 https://doi.org/10.1007/s11258-020-01027-y and Nosko and Embury 2018 https://doi.org/10.1007/s11258-018-0821-7.

The manuscript is very difficult to read and needs to be completely rewritten and shortened to improve readability, flow and clarity. Every element, including captions, needs to be edited and reorganized. Reading through comparisons among sites/treatments is quite cumbersome (see examples on the manuscript). Flow and clarity could be greatly improved by adopting a code or acronym for your site and treatment names (see suggestions in edited manuscript). Flow and length will also be improved by removing site productivity as a factor and presenting plant chemistry data together. I did not provide editorial suggestions for the entire manuscript (see attached), but did so in several sections. Hopefully this will give you an idea of my concerns.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: JHC

Reviewer #2: No

Reviewer #3: No

**********

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Attachment

Submitted filename: Plos OnePhenolics.pdf

pone.0298229.s006.pdf^{(2.6MB, pdf)}

Attachment

Submitted filename: Manuscript - MSG20230306 TRACK CHANGES VERSION.docx

pone.0298229.s007.docx^{(97.4KB, docx)}

PLoS One. 2024 Mar 4;19(3):e0298229. doi: 10.1371/journal.pone.0298229.r002

Author response to Decision Letter 0

6 Aug 2023

All answers to all comments are within the document that I ioloaded, titled: Response to Reviewers - MSG20230804.docx

As you ask for this here as well, I'll copy my text below:

PONE-D-23-06554

Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory

PLOS ONE

Response to Reviewers

Dear Academic Editor,

In this rebuttal letter, I respond to points raised in the review process. First, I repeat the issue/point raised. My response follows after 'Response MSG:', font colour blue, see below.

With regards,

Marcel Schrijvers-Gonlag

Campus Evenstad, Faculty of Applied Ecology, Agricultural Sciences and Biotechnology

Inland Norway University of Applied Sciences, Koppang, Norway

marcel.schrijversgonlag@inn.no

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response MSG: I have studied the style templates in detail and have used these style requirements in the manuscript.

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Response MSG: You ask for correct grant numbers. We used funding from two grants, as stated in the Financial Disclosure. The grant number for the first one is given, but the second grant (from the Extensus Foundation) does not have a grant number. Therefore, no number is provided.

Response MSG: no comment.

Response MSG: I have included the ethics statement in the 'Methods' section.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

Response MSG: I have removed Figure 1 completely.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

Response MSG: The manuscript has been changed according to many reviewer recommendations (under '5. Review Comments to the Author').

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Response MSG: The manuscript has been changed according to many reviewer recommendations (under '5. Review Comments to the Author').

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Response MSG: no comment.

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Response MSG: The manuscript has been changed according to many reviewer recommendations (under '5. Review Comments to the Author').

________________________________________

5. Review Comments to the Author

However there are some minor aspects that need to be taken to be ready to publish

Why no to use another statistic test such as GLM to test interactions between location and soil productivity

Response MSG: The variable Soil productivity has been removed from the manuscript (as suggested by reviewer #3).

Don you think table 1 should be better in supplementary material?

Response MSG: Table 1 is about soil nitrogen. The variable Soil productivity has been removed from the manuscript (as directly suggested by reviewer #3 and indirectly by reviewer #1). This means that also Table 1 has been removed from the manuscript.

I think is more informative to add the letters according to a post hoc test in table two. And why are you adding the column of all treatments together? It is useful??

Response MSG: There is some overlap between Table 2 and Figure 2. I have removed Table 2 and kept the data for all threatments together Table 1 (this is useful information as it indicates the range of all data). The post hoc test letters are in Figure 2 (which is now Figure 1 in the revised manuscript).

Is it on the guidelines or why the figure legends are in the text?

Response MSG: Correct, it says in the PLoS ONE guidelines that the figure legends should be in the text (https://journals.plos.org/plosone/s/figures#loc-how-to-submit-figures-and-captions).

I don’t think that is necessary to add SE and SD from you data every time you describe the results

Response MSG: When presenting mean values, it can be very useful, although not necessary, to give information about either the spread of the data (by presenting the SD) or information about how far the mean is likely to be from the population mean (the 'real value') (by presenting the SE). For each mean value presented I have chosen to include the most appropriate parameter: the SD, the SE, or both.

I think first paragraph of discussion should be a summary of the results, why start with one particular compound?

Response MSG: I have changed the discussion, now it starts with a summary of the results.

Response MSG: Despite an extensive search I was not able to locate papers with other Vaccinium species that addresses the particular topic that is discussed in this paragraph, therefore I used papers with Mountain birch and Mopane who do address the particular topic instead. To satisfy some reviewers' requests, I have removed this particular text.

The supplementary file provided by Reviewer #1 contained many textual suggestions and some questions. I have answered the questions in the text and changed the manuscript accordingly, when appropriate. Some changes were not necessary and more a matter of taste, in these cases I often did not change my own words. As the text concerning the variable Soil productivity has been removed in the new version of the manuscript, many comments about this part can be neglected.

Response MSG: I have removed the Protein Competition Model and all text concerning proteins from the Introduction chapter and mentioned the PCM briefly in the Discussion chapter.

Further, the conclusion that this may be due to clonality is likewise interesting, but was not tested. Did you look at C:N ratios in clonal ramets connected to experimental ramets, for example, to see whether there may be a flux or allocation of resources to damaged plants.

Response MSG: Clonality is only mentioned in the Discussion section, and is not mentioned in the Conclusion section. We did not look at C:N ratios in clonal ramets as this was not the goal of our study.

Additionally, why was the foliar content not measured? Especially considering you had a defoliation treatment, one might expect differences to be most apparent in leaves. Authors should justify why this was.

Response MSG: We did not measure foliar content as our study focused on the influence of simulated herbivory (on leaves, but also more severe herbivory on shoots) on phenolic content and C:N ratio in annual shoots. One of the reasons that we focused on annual shoots instead of leaves was that many other studies have focused on leaves but studies on annual shoots were scarce.

Response MSG: We did measure several morphological properties of all bilberry ramets, including biomass of annual shoots, but these data will be used in another manuscript. In the reviewed manuscript we focus on chemical responses following herbivory, in a future manuscript we will focus on morphological responses following herbivory.

Although the takeaway message is rather simple, I found the MS slightly challenging to read. And was distracted by a lot of possibilities and details that were not tested in the experiments. Perhaps if the paper was reframed and simplified to highlight what was tested, as opposed to what was not (all of these trade-off theories) it would be easier to follow. For example, your first prediction is not even possible to test, because you don’t detect any gallic acid-derived phenolics at all. As such, it doesn’t make much sense to include it in the MS. Your first prediction might instead be something like ‘determine whether bilberry has these compounds at all, and compare them with Phe-derived phenolics’. Then you might be able to contextualize this in the protein competition model. However, because they are not present, this is of course not possible.

Response MSG: I have removed the first prediction and the Protein Competition Model from the Introduction chapter and mentioned the PCM briefly in the Discussion chapter. Furthermore, I have adjusted the text at many places in order to make it easier to read.

I did not leave specific, detailed comments, as lines were not numbered. In subsequent drafts I would recommend adding line numbers.

Response MSG: Line numbers were present in the manuscript but unfortunately disappeared just for submission (possibly due to a Word update). I found out about this just after submission and contacted the PLoS ONE editor instandly. I got the following reply: '... I believe the reviewers will find their way to address targeted comments. If they encounter problems regarding line numbering, I will get back to you.'

In the revised manuscript I have added line numbers.

Response MSG: The variable Soil productivity has been removed from the manuscript. Also, the two groups that were analysed separately in the reviewed manuscript, have been merged into one dataset, and all analyses have been adjusted accordingly, as suggested by reviewer #3. Furthermore, I have adjusted the text at many places in order to make it easier to read.

Present the plant chemistry data (phenols, tannins, N, C, and C/N) together in one Table or Figure, so that the reader can better visualize possible relationships among these variables (for example, see Nosko et al 2020, https://doi.org/10.1016/j.foreco.2019.117839).

Response MSG: I have taken over this suggestion and many chemistry data (tannins, all phenolics, C, N, C/N) are now in one table (Table 1).

In your discussion, you can then focus on browsing treatments as they affect the relationships among plant chemistry variables. In the absence of such relationships, some inspiration might be obtained from Warbrick et al 2020 https://doi.org/10.1007/s11258-020-01027-y and Nosko and Embury 2018 https://doi.org/10.1007/s11258-018-0821-7.

Response MSG: I have changed the discussion according to several recommendations from the reviewers.

Response MSG: I have adjusted the text at many places in order to make it easier to read. For this, the many adjustments made by Reviewer #1 and Reviewer #3 have been very useful.

Reading through comparisons among sites/treatments is quite cumbersome (see examples on the manuscript). Flow and clarity could be greatly improved by adopting a code or acronym for your site and treatment names (see suggestions in edited manuscript).

Response MSG: The suggested codes/acronyms in the edited manuscript have been used in the revised manuscript.

Flow and length will also be improved by removing site productivity as a factor and presenting plant chemistry data together.

Response MSG: The variable Soil productivity has been removed from the manuscript and most chemistry data are now in one table (Table 1).

I did not provide editorial suggestions for the entire manuscript (see attached), but did so in several sections. Hopefully this will give you an idea of my concerns.

Response MSG: All suggestions for improvement have been considered. As with Reviewer #1, I have changed the manuscript accordingly, when appropriate.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: JHC

Reviewer #2: No

Reviewer #3: No

Attachment

Submitted filename: Response to Reviewers - MSG20230804.docx

pone.0298229.s008.docx^{(34.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0298229.r003

Decision Letter 1

Branislav T Šiler

12 Dec 2023

PONE-D-23-06554R1Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory.PLOS ONE

Dear Dr. Schrijvers-Gonlag,

==============================Reviewer #2 raised several very important concerns regarding the tradeoff existing in changing herbivory regimes between defense and metabolic processes. Please take into a careful consideration their points, which can aid in further improvement of the manuscript quality. Furthermore, a visual presentation of the study setup would be much helpful.==============================

Please submit your revised manuscript by Jan 26 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: This is my second time reviewing this paper, and while I appreciate that the authors have made substantial revisions to their MS, which have certainly increased the overall quality and clarity of the paper, I still have some concerns regarding the data presentation and the claims made. Namely, there is substantial mention of growth, for example, lines 60, 369 and 459. While I understand authors may want to use biomass data (which they have admittedly collected) for a second MS, I find that it is an important piece of data that would make their claims and discussions more robust and compelling. Especially if the aim is to, at least in part, understand the tradeoffs between defense and other metabolic processes across variable herbivory regimes. For example, you can not say for certain that plants are prioritizing growth more or less between different defoliation treatments because you have no metric of growth (total biomass, compensatory growth). To me this is extremely relevant. One scenario I could think of is that the loss of biomass from mild herbivory treatments is effectively inconsequential for plant fitness and thus responses are not obvious. When biomass loss becomes too great, plants must begin to divert resources from other pools to compensate for biomass losses. It would be interesting to see the differences in growth after herbivory treatments, which would determine if severely damaged plants actually put on more biomass then more moderately damaged plants, or if simply the amount of resources are so low that these severely damaged plants not only produce less phenolics, but also less growth and thus metabolize less, generally. Also, while the C and N concentrations may remain stable across treatments, the total pool of each that plants have access to is not consistent; in this case it is likely a function of biomass. So the amount of carbon severely damaged plants have access to should be less then mildly-damaged plants if there is a biomass reduction.

Additionally, I find the entire conclusion section quite difficult to follow. What exactly is the takeaway message?

Finally, perhaps a diagram of the plant treatments would be helpful. It is difficult to determine exactly how your treatments were conducted. As such, I can't help but wonder if some of your results could be an artifact of some concentration/dilution effect by sampling different numbers of stems/ramets between treatments, that is more severe with increasing herbivory. A clear outline (visual) could help to assuage these concerns.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

**********

PLoS One. 2024 Mar 4;19(3):e0298229. doi: 10.1371/journal.pone.0298229.r004

Author response to Decision Letter 1

19 Jan 2024

6. Review Comments to the Author

Response MSG: we have been in contact with PLoS ONE by email (12 January 2024), as we cannot meet this point. The reply I got from PLoS ONE is below (received on 15 January 2024):

"I agree that the suggested morphometric evaluation would be an

exhausting job and further delay the manuscript publication. Although I

strongly support the Reviewer #2's demand for additional experimental

assessment, which would remarkably contribute the study

comprehensiveness, I find the manuscript in this form contains a fair

dose of novel scientific information that candidate it for acceptance

for publication if other reviewer's requirements are to be met. However,

I urge the authors to properly discuss in the text the limitations of

the present findings in light of Reviewer #2's comments."

Best regards,

Branislav

Response MSG: Therefore, I have added a paragraph in the Discussion section, called ‘Defense and other metabolic processes’, to discuss this issue.

Additionally, I find the entire conclusion section quite difficult to follow. What exactly is the takeaway message?

Response MSG: The conclusion section has been changed, to clarify our intention.

Response MSG: I have added a new Figure 1, which illustrates the study design.

Attachment

Submitted filename: Response to Reviewers - MSG20240119.docx

pone.0298229.s009.docx^{(24.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0298229.r005

Decision Letter 2

Branislav T Šiler

22 Jan 2024

Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory.

PONE-D-23-06554R2

Dear Dr. Schrijvers-Gonlag,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0298229.r006

Acceptance letter

Branislav T Šiler

23 Feb 2024

PONE-D-23-06554R2

PLOS ONE

Dear Dr. Schrijvers-Gonlag,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

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If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

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on behalf of

Dr. Branislav T. Šiler

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Annual shoots and random selection.

(PDF)

pone.0298229.s001.pdf^{(89KB, pdf)}

S2 File. Standard reference curve and tannin color test.

(PDF)

pone.0298229.s002.pdf^{(228.9KB, pdf)}

S3 File. Quantifying phenolics using HPLC.

(PDF)

pone.0298229.s003.pdf^{(154.2KB, pdf)}

S1 Table. Response factors.

(PDF)

pone.0298229.s004.pdf^{(100.4KB, pdf)}

S2 Table. Phenolic concentrations after simulated herbivory.

(PDF)

pone.0298229.s005.pdf^{(522.5KB, pdf)}

Attachment

Submitted filename: Plos OnePhenolics.pdf

pone.0298229.s006.pdf^{(2.6MB, pdf)}

Attachment

Submitted filename: Manuscript - MSG20230306 TRACK CHANGES VERSION.docx

pone.0298229.s007.docx^{(97.4KB, docx)}

Attachment

Submitted filename: Response to Reviewers - MSG20230804.docx

pone.0298229.s008.docx^{(34.3KB, docx)}

Attachment

Submitted filename: Response to Reviewers - MSG20240119.docx

pone.0298229.s009.docx^{(24.1KB, docx)}

Data Availability Statement

The datasets and scripts used for the presented analyses are stored in the DataverseNO database and available at https://doi.org/10.18710/U8S3XX.

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PERMALINK

Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory

Marcel Schrijvers-Gonlag

Christina Skarpe

Riitta Julkunen-Tiitto

Antonio B S Poléo

Roles

Abstract

Introduction

Methods

Study area

Study design

Field treatments

Fig 1. Study design.

Bilberry sampling

Chemical analyses

Bilberry shoots

Shoot tannins

Shoot phenolics

Shoot carbon and nitrogen

Statistical analyses

Fig 2. Tannin concentration in bilberry annual shoots after simulated herbivory.

Fig 5. Individual phenolic concentration in bilberry annual shoots after simulated herbivory.

Results

Shoot tannins

Table 1. Tannins, phenolics, carbon, nitrogen, and C/N in bilberry annual shoots after simulated herbivory.

Shoot phenolics

Fig 3. HPLC chromatogram of phenolics in bilberry annual shoots.

Fig 4. Total phenolic concentration in bilberry annual shoots after simulated herbivory.

Table 2. Differences in individual phenolic concentration in bilberry annual shoots between simulated herbivory treatments.

Shoot carbon and nitrogen

Discussion

Shoot tannins and total phenolics

Shoot individual phenolics

Shoot carbon and nitrogen

Study design

Defense and other metabolic processes

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Branislav T Šiler

Roles

Author response to Decision Letter 0

Decision Letter 1

Branislav T Šiler

Roles

Author response to Decision Letter 1

Decision Letter 2

Branislav T Šiler

Roles

Acceptance letter

Branislav T Šiler

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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