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Rinnakkaistallenteet Luonnontieteiden ja metsätieteiden tiedekunta
2018
A Seven-Year Study of Phenolic
Concentrations of the Dioecious Salix myrsinifolia
Nissinen, Katri
Springer Nature
Tieteelliset aikakauslehtiartikkelit
© Springer Science+Business Media, LLC, part of Springer Nature 2018 All rights reserved
http://dx.doi.org/10.1007/s10886-018-0942-4
https://erepo.uef.fi/handle/123456789/6753
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A SEVEN-YEAR STUDY OF PHENOLIC CONCENTRATIONS
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OF THE DIOECIOUS Salix myrsinifolia
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*KATRI NISSINEN1
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VIRPI VIRJAMO1
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LAURI MEHTÄTALO2
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ANU LAVOLA1
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ANU VALTONEN1
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LINE NYBAKKEN3
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RIITTA JULKUNEN-TIITTO1
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1Department of Environmental and Biological Sciences, University of Eastern Finland (UEF), 80101, Joensuu, Finland
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2School of Computing, University of Eastern Finland, 80101, Joensuu, Finland
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3Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, 1432
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Ås, Norway
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*Correspondence: Katri Nissinen, e-mail: katri.nissinen@uef.fi, Telephone +358 50 381 9326
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ORCIDS – Katri Nissinen 0000-0002-6744-2773, Virpi Virjamo 0000-0002-2967-4813, Anu Valtonen 0000-0003-
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1532-1563
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Abstract–In boreal woody plants, concentrations of defensive phenolic compounds are expected to be at a high level
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during the juvenile phase and decrease in maturity, although there is variation between plant species. Females of
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dioecious species, like most of the Salicaceae, are expected to invest their resources in defense and reproduction, while
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males are expected to be more growth-oriented. We studied age- and sex-dependent changes in leaf and stem phenolics,
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and in height and diameter growth in a dioecious Salix myrsinifolia plants over a seven-year time period. In addition,
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we registered flowering as well as rust damage in the leaves. From the first year and throughout ontogenetic
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development from juvenile to adult phases, there was no significant change in the concentrations of any of the studied
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compounds in the leaves of S. myrsinifolia. In the stems, the concentrations of six out of 43 identified compounds
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decreased slightly with age, which may be partly explained by dilution caused by the increment in stem diameter with
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age. The fairly steady chemistry level over seven years, accompanied by moderate genotypic phenolic variation,
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indicates important roles of chemical defenses against herbivory for this early-successional species.
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Key Words–Salix myrsinifolia, Dark-leaved willow, Melampsora, Sexual differences, Phenolic compounds.
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INTRODUCTION
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In plants, phenolic compounds, such as salicylates, flavonoids, phenolic acids and condensed tannins, function as
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defensive compounds against various herbivores, as well as pathogens (Barbehenn and Constabel 2011; Boeckler et al.
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2011). Flavonoids are also acknowledged to have antioxidant properties e.g. in photoprotection (Agati et al. 2012, 2013;
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Ferreyra et al. 2012). Defense levels vary across ontogenetic phases (Barton and Koricheva 2010; Boege and Marquis
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2005; Donaldson et al. 2006). Although there is variation between plant species, the concentration of different phenolic
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compounds generally increases during the juvenile seedling stage of boreal woody plants (Barton and Koricheva 2010;
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Donaldson et al. 2006). After the seedling phase, during the period of intensive growth, the concentration of total
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phenolics often remains at constant level (Barton and Koricheva 2010). Later, during the reproductive phase, the
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concentration usually decreases as resources are directed to flowering (Boege and Marquis 2005). Especially in boreal
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areas with harsh winters, phenolic concentrations in woody species have been found to be lower in mature plants than in
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juvenile ones (Barton and Koricheva 2010). Consequently, mature plants have been preferred for feeding by mammals
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and birds (Boege and Marquis 2005, Tahvanainen et al. 1985). Proportions of different compounds and compound
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groups may change during ontogeny, and as an example, salicylates can be replaced by other phenolic compounds such
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as flavonoids and condensed tannins as the plant develops (Julkunen-Tiitto 1989a).
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Females and males of dioecious species differ in their morphology, physiology, and defensive strategies
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(Obeso 2002). According to ecological theory, the females of dioecious species should invest more in reproduction and
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defense, whereas males are more growth-oriented (Obeso 2002). Experimental data are both in favor of and against this
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theory (Nybakken and Julkunen-Tiitto 2013; Randriamanana et al. 2015a, 2015b; Robinson et al. 2014). Differences
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between sexes in their adaptation to natural habitats may cause female- or male-biased sex ratios (Munné-Bosch 2015).
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Male plants of different plant species are often more susceptible to herbivory (Cornelissen and Stiling 2006, Ågren et al.
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1999). This difference is related to sexual differences in chemical composition, leaf morphology, vegetative phenology
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as well as in the resulting nutrient content of the plants (Ågren 1999).
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The rapid-growing Salix myrsinifolia is a dioecious species native to Europe and western Siberia. S.
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myrsinifolia has variable growth habits, varying from a small-sized shrub to a small tree, thriving in many different
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humid habitats (Cronk et al. 2015; Skvortsov 1999). As Salicaceae species in general, S. myrsinifolia is a dioecious
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species (Skvortsov 1999). It is, as most of the Salix species, sexually female-biased 2:1 (Hughes 2010). In a three-year
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field study, males of S. myrsinifolia had higher diameter and showed stronger growth response to enhanced temperature
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during the first two years than did females, while females had more chlorogenic acids in leaves during the third year
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compared to males (Nybakken et al. 2012; Randriamanana et al. 2015a). In a greenhouse experiment, where plants grew
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in optimal conditions compared to those in field studies, female plants had clearly higher concentrations of most of the
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studied phenolic compounds in twigs than males (Nybakken and Julkunen-Tiitto 2013).
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Like most species belonging to the Salicaceae family, S. myrsinifolia contains high concentrations of
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salicylates in its leaves and stems (Boeckler et al. 2011; Julkunen-Tiitto 1989a; Julkunen-Tiitto and Virjamo 2017). The
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salicylates differ both qualitatively and quantitatively between Salicaceae species and among genotypes within the
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species (Förster et al. 2008; Heiska et al. 2008; Nybakken et al. 2012; Nyman and Julkunen-Tiitto 2005). Usually,
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salicylates and their degradation products repel generalist herbivores (e.g. Osier and Lindroth 2001; Pentzold et al.
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2014; Volf et al. 2015). On the other hand, Salix specialist herbivores are attracted by the high concentration of
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salicylates (Tahvanainen et al. 1985). Some specialist species can even benefit from salicylates and use the glucose
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moiety as an energy source and the salicylaldehyde moiety in their own defense systems (Boeckler et al. 2000, Rowell-
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Rahier and Pasteels 1986). By local outbreaks, these specialists can cause high seasonally varying stress on Salix plants
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(Björkman et al. 2000). In addition to herbivores, pathogens, for example the Melampsora-species, may be detrimental
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to the growth of Salix species (Dawson and McCracken 1994). Increased concentrations of some phenolic compounds
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have been reported to decrease the infections of Melampsora-rust in S. myrsinifolia and S. triandra (Hakulinen 1998,
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Hakulinen et al. 1999, Hjältén et al. 2007).
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Concentrations of salicylates in many Salicaceae species change during they ontogeny (Nissinen et al.
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2017, Nybakken et al. 2012, Randriamanana et al. 2015a). A high level of phenolics during the seedling phase is an
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ancestral state, which some species from the subgenus Salix share with Populus (e.g. Julkunen-Tiitto 1989b). Populus is
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a sister genus of Salix (Skvortsov 1999, Wu et al. 2015). For example, in P. tremuloides salicylates decreased with age,
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while condensed tannins increased (Donaldson et al. 2006). On the other hand, in S. myrsinifolia, the salicylate level
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can be at a high level even in mature plants (Julkunen-Tiitto 1989b). This may be one sign of S. myrsinifolia being a
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derived species. However, phylogeny of the genus Salix is still under study (e.g. Lauron-Moreau et al. 2015, Wu et al.
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2015). Skvortsov (1999) divided the genus Salix into three subgenera; Salix, Chamaetia and Vetrix, whereas e.g.
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Ghahremaninejad et al. (2012), supports a unification of two subgenera, Chaemaetia and Vetrix, into one, based on leaf
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morphological characters. S. myrsinifolia belongs to the subgenus Vetrix, which includes some of the most derived Salix
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species (Lauron-Moreau et al. 2015, Skvortsov 1999).
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Here we present a seven-year field study on the ontogenetic, age-related and sex-dependent patterns of
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phenolic compounds, and on rust infestation of S. myrsinifolia. To our knowledge, this is the first long-term study on
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the development of detailed leaf phenolic profiles over the ontogenetic phases in any tree species. In addition, we
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studied changes in stem phenolics, height and diameter growth, and in flowering. Based on phytochemistry levels
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measured from young (Nybakken et al. 2012) and adult S. myrsinifolia plants (Julkunen-Tiitto 1989b), we did not
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expect S. myrsinifolia to undergo great changes over age in leaf and stem phenolics, neither qualitatively nor
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quantitatively. On the other hand, we expected to find sex-related differences in leaf and stem phenolics, and also in
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flowering and growth. We hypothesized that (1) males would be more growth-oriented, while females would invest
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more in defensive compounds and reproduction. (2) Age would not influence the level of secondary compounds in the
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leaves or stems of S. myrsinifolia. (3) Age would not influence the severity of rust infestation in the leaves, which we
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expect to depend mainly on varying weather conditions between years.
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METHODS AND MATERIALS
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Plant Material and Experimental Design. In May 2007, we collected approx. 25 cm long cuttings from eight female and
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nine male genotypes from the second-year-shoots of S. myrsinifolia plants. The samples were obtained from wild
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populations sampled at nine different locations more than 8 kilometers apart in Eastern Finland. At each location, we
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sampled a male and a female genotype, keeping a minimal distance of 500 m between the sampled individuals. The
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same spring, we planted 40 cuttings of each genotype randomly in eight rows in an abandoned hay field in Luikonlahti
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(62°54'N, 28°40'E) in Eastern Finland.
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Growth Measurements and Sampling. Height and diameter growth measurements and sampling of leaves were done
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every year at the end of the growing season, starting from the year of planting (Table S1). Sampling of stems was done
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after the growing season (Table S1). The stem samples from 2009–2011 could not be used because the freezer, where
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the samples were kept, broke down. Diameter was measured at the root collar, 1 cm above the ground level, using a
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digital caliper. To detect possible differences between sexes in the starting time of flowering, we counted the number of
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flowering plants and the number of the catkins in individual plants in 2009 and 2010.
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Two to six of the youngest fully expanded leaves of the longest shoot were collected for chemical
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analyses as well as for assessing leaf area, leaf dry weight, rust infection severity and herbivory damage. Leaves were
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collected from six to ten individual plants of eight female genotypes and nine male genotypes per year (total 150–153
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individuals per year, full total 1058). The leaves were air-dried in paper bags containing drying material (about 2 g of
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silicagel, Oy FF-Chemicals Ab, Haukipudas) in a drying room at a relative humidity of 10 % (at room temperature) for
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12 h. The dried leaves were weighed and their area was measured with a portable leaf area meter LI-3000C (LI-COR,
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Lincoln, NE, USA). Specific leaf area (SLA, cm2 g-1 dw) was calculated by dividing leaf area by leaf dry weight. The
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number of Melampsora-rust infection uredinia spots per leaf area was calculated by microscopic examination of the
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same leaves that were used for chemistry analysis, in all the years 2007–2013. 10 cm long stem internode parts were
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collected from a side shoot of three to ten individual plants of each genotype per year (total 72–143 individuals per
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year, full total 494). Stem parts were kept at -20 °C before chemical analyses. Stems of one female genotype and four
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male genotypes were not included in the study in 2007, as the number of plant individuals with side branches was too
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low in these genotypes.
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Extraction and Analysis of Leaf and Stem Phenolics. Leaf phenolics were extracted according to earlier published
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methods (Nybakken et al. 2012). In short, approximately 6 mg of leaf discs was cut from the leaf blades of the sampled
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2–6 leaves, avoiding leaf veins, mixed and homogenized for 30 s in 600 μl ice-cold 100 % methanol (MeOH) at 5500
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rpm using a Precellys-homogenizer (Bertin Technologies, France). The samples were incubated in an ice bath for 15
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min and centrifuged at 16700 g for 3 min at 4 °C (Eppendorf Centrifuge 5415 R, Hamburg, Germany). Supernatants
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were collected immediately after centrifugation. The sample residues were re-extracted three times. Supernatants from
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extractions were pooled and evaporated to dryness in a vacuum centrifuge (Eppendorf 270 Concentrator, Hamburg,
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Germany) at +45 °C and stored at -20 °C. The pellets were dried in a fume hood at room temperature, stored at -20 °C,
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and later used for quantification of the MeOH-insoluble condensed tannins.
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Before extraction of stem phenolics, buds and a 2 cm piece from the stem tips were removed, and a 2.5
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cm piece of stem was cut for chemical analyses. In addition, a 3 cm piece of the stem was cut for dry weight
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measurement (dried in an oven at 105 °C for 48 hours). The 2.5 cm piece of stem was cut up into small pieces in a cold
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room (4 °C) and they were put on ice. 20–25 mg of the stem pieces were homogenized for 30 s at 5500 rpm using a
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Precellys-homogenizer (Bertin Technologies, France). After homogenization, the stem phenolics were extracted
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similarly to the method used for leaf phenolics.
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For analyses of leaf and stem phenolics, the dried leaf samples were re-dissolved in 1.2 ml
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MeOH:MilliQ water (1:1), while the stem samples were re-dissolved in 1.0 ml MeOH:MilliQ water (1:1). Both sample
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types were run on an HPLC-DAD system that consisted of a binary pump, a vacuum degasser, an autosampler with a
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thermostat, a column oven and a diode array detector. A column Zorbax SB-C18 (4.6 x 75 mm, particle size 3.5 μm) was
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used. The eluents used were A (1.5 % tetrahydrofuran + 0.25 % orthophosphoric acid in Milli-Q ultrapure water) and B
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(100 % MeOH). The gradient for eluent A was as follows: 0–5 min 100 %, 5–10 min 100–85 %, 10–20 min 85–70 %,
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20–40 min 70–50 %, 40–50 min 50 %, 50–52 min 50–0 %. The column temperature was 30 °C, the flow rate 2 ml min-
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1, and the injection volume 20 μl.
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The salicylates and other glucosides were quantified at 270 nm, flavonoids and phenolic acids at 320
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nm. The concentrations of compounds (mg g-1 dw) were calculated according to the following standards: salicin
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(Sigma-Aldrich Finland Oy, Helsinki, Finland) for salicin, salicyl alcohol and diglucoside of salicyl alcohol; salicortin
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(Sigma-Aldrich Finland Oy, Helsinki, Finland) for salicortin, disalicortin, HCH-salicortin derivatives 1 and 2;
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tremulacin (Apin Chemicals, Abingdon, UK) for tremulacin; (+)-catechin (Fluka Chemie AG, Buchs, Switzerland) for
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(+)-catechin; quercetin 3-galactoside (Apin Chemicals, Abingdon, UK) for quercetin 3-galactoside, quercetin
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diglucoside, quercetin 3-glucoside, quercetin glycoside derivative, quercetin 3-arabinoside, quercetin 3-rhamnoside and
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quercetin rhamnetin derivative; myricetin 3-rhamnoside (Apin Chemicals, Abingdon, UK) for myricetin galactoside and
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myricetin glucoside+glucuronide; luteolin 7-glucoside (Carl Roth, Karlsruhe, Germany) for luteolin 7-
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glucoside+quercetin, luteolin glycoside derivatives 1 and 2, monomethyl luteolin 7-glucoside and luteolin derivative;
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eriodictyol 7-glucoside (Carl Roth, Karlsruhe, Germany) for eriodictyol aglycone derivatives 1 and 2; apigenin 7-
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glucoside (Carl Roth, Karlsruhe, Germany) for apigenin 7-glucoside; kaempferol 3-O-glucoside (Extrasynthése, Genay,
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France) for coumaroyl-astragalin; chlorogenic acid (Sigma-Aldrich Finland Oy, Helsinki, Finland) for neochlorogenic
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acid, chlorogenic acid, and chlorogenic acid derivatives 1, 2, 3, 4 and 5; cinnamic acid (Sigma-Aldrich Finland Oy,
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Helsinki, Finland) for p-OH-cinnamic acid derivatives 1, 2, 3, 4 and 5; picein (Extrasynthése, Genay, France) for
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picein; triandrin (from Prof. Beat Meier, Zürich, Switzerland) for triandrin; salidroside (from Prof. Beat Meier, Zürich,
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Switzerland) for rosavin derivative; salireposide (from Prof. Beat Meier, Zürich, Switzerland) for salireposide.
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The acid butanol assay (Hagerman 2011) was used to quantify condensed tannin concentration.
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Concentrations of MeOH-soluble condensed tannins were determined from the re-dissolved MeOH:water extracts used
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for HPLC-runs and MeOH-insoluble condensed tannins were determined from the dried pellet samples from MeOH
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extractions. Purified crude tannin extracts from S. myrsinifolia leaves and stems were used as standards.
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Statistical analyses. The effects of age and sex on the concentrations of leaf and stem total salicylates, total flavonoids,
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total phenolic acids, total condensed tannins, soluble and insoluble condensed tannins and individual phenolic
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compounds, height and basal diameter, leaf weight, leaf area and specific leaf area (SLA), for genotype i, plant
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individual j and year k were analyzed using the linear mixed-effect model
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𝑦𝑦𝑖𝑖𝑖𝑖𝑖𝑖=𝜷𝜷′𝒙𝒙𝒊𝒊𝒊𝒊𝒊𝒊+𝑢𝑢𝑖𝑖(1)+𝑢𝑢𝑖𝑖(2)𝑡𝑡𝑖𝑖𝑖𝑖𝑖𝑖+𝑣𝑣𝑖𝑖𝑖𝑖(1)+𝑣𝑣𝑖𝑖𝑖𝑖(2)𝑡𝑡𝑖𝑖𝑖𝑖𝑖𝑖+𝑝𝑝𝑖𝑖+𝑒𝑒𝑖𝑖𝑖𝑖𝑖𝑖 (1)
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where the fixed part β’xijk includes the fixed predictors (age, sex and their interaction), ui(1) and ui(2) are random normally
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distributed intercept and slope coefficient of age (tijk) at the genotype level and vij(1) and vij(2) are the random intercept
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and slope at the plant individual level within genotype, pk is the random effect for calendar year and eijk is the residual
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error for plant individual j in genotype i in year k. The age was coded as a continuous predictor, with an additional
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binary predictor for the first year, when the plants were still at the juvenile phase.
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In addition, we analyzed flowering and leaf rust infection severity by the linear mixed effect model.
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Flowering and leaf rust infection severity were clearly a mixture of two random processes: i) the effects on presence
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and absence of catkins or severe rust infection and ii) the effects on magnitude of the variable of interest. These data
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were therefore analyzed in two parts. First, when analyzing the presence of catkins and the occurrence of severe
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infection (≥ 1000 rust spots per leaf), we used a binary logistic mixed-effect model. In this model, the fixed part and
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random effects were specified as in the previously introduced linear mixed-effect model (1). Second, we used this linear
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mixed-effect model to analyze the number of catkins for those plants where these occurred and the number of rust spots
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for leaves with non-severe rust infection.
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The model selection was based on the conditional F tests and t tests, and on Likelihood ratio (Chi2)
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tests, if F tests or t tests were not possible (Pinheiro and Bates 2000). The model fit was evaluated graphically and when
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needed, a logarithmic (ln(x), ln(x+0.1) or ln(x+1)) transformation (Table S2) was used in the response variable for
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testing purposes. For each variable, we selected the transformation that visually showed the most homoscedastic
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residuals of the variable in question. However, the final parameter estimates are reported using a model fitted into the
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original scale, to enable meaningful interpretation (Table S2). The approach was selected because the coefficients and
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predictions from a model fitted in the original scale are unbiased, even though not fully efficient, whereas back-
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transformed predictions from a transformed model would be biased. The statistical analyses were conducted using R
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version 3.3.1 (R Core Team 2016) with the packages lme4 (Bates et al. 2015) and lmerTest (Kuznetsova et al. 2016) in
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RStudio 0.99.903 (© 2009–2016 RStudio, Inc., Boston, USA) and IBM® SPSS® Statistics 22 (Armonk, NY, USA).
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We did a graphic vector analysis (GVA) (Figure 1) to study the effects of age on the different phenolic
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group levels; salicylates, flavonoids, phenolic acids and condensed tannins, in leaves of S.myrsinifolia (Haase and Rose
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1995, Koricheva et al. 1999). The relative concentration and content values in GVA were calculated by using the
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concentration (mg g-1) and content (mg) values of the phenolic group in the first-year plants as a reference. We plotted
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the relative values of concentration (y) and content (x) of different phenolic compound groups in relation to leaf dry
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weight of the leaves (z) used for phenolic analysis. In a GVA plot, an excess synthesis of a certain phenolic group
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compared to the first year is revealed when both its relative content and concentration are increasing. Decreased relative
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content and concentration implies reduced synthesis in the phenolic compound group of interest. A concentration effect
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in the tissue is revealed if relative content decreases while relative concentration increases. On the other hand, an
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increase in relative content and a decrease in relative concentration indicates dilution. The relative values used in a
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GVA plot were calculated as studied year mean/first year mean x 100. The GVA plots were drawn using SigmaPlot
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13.0 (Systat Software, Inc., Chicago, IL, USA). Because we did not collect the stem biomass samples, we did not have
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stem biomass data for calculation of the relative content of phenolics for the GVA. Therefore, we did not analyze stem
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phenolics with the GVA method.
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We used a principal component analysis (PCA) to get an overview of the total leaf and stem phenolics
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dataset (Figure S3, Figure S4). In the PCA we plotted all the observations according to all 33 different leaf and 43
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different stem phytochemical compound concentrations to visualize the relationships of different genotypes in seven-
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year time period. Sex, genotype and year were set as secondary observations identifiers. In addition, we scored the
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leaves based on the amount of rust pustules on them (0–299=1, 300–799=2, >800=3). This rust score data was set as a
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secondary observation identifier in the PCA to visualize the effect of rust infection severity on leaf phenolics. The PCA
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was conducted in the SIMCA-P+-progman (Umetrics AB, Sweden). Furthermore, to obtain the proportion of variation
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in leaf phenolic profiles explained by rust infection, age, sex and genotype, we used a permutational ANOVA-model
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(PERMANOVA+ in PRIMER-E-software, version 6) (Anderson et al. 2008) to fit a hierarchical design for our data
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(type III sums of squares and using Bray-Curtis as a measure of similarity). In PERMANOVA, rust infection and age
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were added as continuous variables, sex as a fixed factor with two levels, genotype nested in sex and individual nested
232
in genotype as random factors.
233 234
RESULTS
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Leaf and Stem Phenolics. In the leaves of S. myrsinifolia, we detected and quantified 31 different low molecular weight
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phenolic compounds, in addition to soluble and insoluble high molecular weight condensed tannins (proanthocyanidins)
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(Table 1). Salicylates were the main group of low molecular weight phenolics, representing 72 % of their total
239
concentrations in the leaves (Figure 2). Other groups were phenolic acids and flavonoids, representing shares of 19 %
240
and 9 %, respectively. Among the salicylates, salicortin and HCH-salicortin were the most abundant. Chlorogenic acids
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were the most abundant phenolic acids, while quercetin 3-galactoside and (+)-catechin were the most abundant
242
flavonoids.
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In the stems of S. myrsinifolia the identified number of different compounds, 43, was higher than in the
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leaves (Table 2). Salicylates formed the most abundant low molecular weight phenolic group also in the stems.
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Salicortin and salicin were present in the highest quantities. The stems contained phenolic glycosides not detected in the
246
leaves. Main phenolic glycosides were picein and a rosavin derivative. In contrast to the leaves, the stems had higher
247
concentrations of flavonoids than of phenolic acids, but the most abundant compounds were the same in both of these
248
phenolic groups in leaves and stems.
249
We found almost no significant developmental trends in the concentrations of the major phenolic
250
groups or of the individual phenolic compounds in the leaves of S. myrsinifolia. In the early development of the S.
251
myrsinifolia plants, between the first and the second year, we detected a significant increase in the concentration of only
252
three minor compounds of the analyzed leaf phenolics; in HCH-salicortin 2 (t(4.012)=-9.028 P<0.001), in monomethyl
253
luteolin 7-glucoside (t(3.997)=-3.453 P<0.05) and in luteolin 7-glucoside+quercetin glycoside (t(3.995)=-2.968 P<0.05)
254
(Table S2). No other significant effect of age was detected on leaf phenolics. The results of GVA analysis showed
255
increase in the concentrations of total phenolic acids and total salicylates only between 2007 and 2008. There was no
256
clear trend in the following years (Figure 1; Table S2). When visualizing the whole leaf individual phenolic compound
257
dataset in a PCA plot, years did not differ from each other (Figure S3). Based on PERMANOVA model, gneotype
258
explained the largest part of the variation in compositions of leaf phenolics (40.7%), while the proportion of variation
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explaned by age was only 10.2% (Table 3).
260
We detected some developmental trends in the stem phenolics of S. myrsinifolia. Concentrations of six
261
out of the 43 low molecular weight phenolic compounds decreased significantly with increasing age (Table 2; Table
262
S2). These were salicin (F(2,29.880)=60.616 P<0.001), HCH-salicortin1 (χ2(2)=14.207 P<0.001), monomethyl 7-glucoside
263
(χ2(2)=15.061 P<0.001), methyl luteolin 7-glucoside (χ2(2)=7.116 P<0.05), chlorogenic acid derivative2
264
(F(2,27.069)=19.057 P<0.001) and salireposide (χ2(2)=9.830 P<0.01) (Table 2; Table S2). Two compounds, salicin
265
(t(422.2)=7.450 P<0.001) and chlorogenic acid derivative2 (t(363.9)=-3.823 P<0.001), decreased between the first and the
266
second year. In addition, phenolic glycosides as a group (χ2(2)=7.788 P<0.05) and MeOH-insoluble condensed tannins
267
(χ2(2)=9.916 P<0.01) decreased with age in stems. Furthermore, though not significant, there was a decreasing trend in
268
the concentrations of all the low molecular weight phenolic groups and most of the individual compounds in the stems
269
(Table 2; Table S2; Figure S4).
270
Generally, we found only minor differences in both leaf and stem phenolics between male and female
271
genotypes. In females, we detected significantly higher concentrations of the chlorogenic acid derivative2 (t(15.074)=-
272
2.560 P<0.05) in the leaves and of neochlorogenic acid (t(15.025)=-2.24 P<0.05) in the stems. On the other hand, the
273
concentrations of total flavonoids, (+)-catechin and condensed tannins in the leaves tended to be higher in males (Table
274
1; Table S2). The similarity in the phenolic profiles of the sexes can be seen in the PCA plot (Figure S3), and is also
275
supported by the results of the PERMANOVA model (Table 3).
276
In contrast to low sexual differences, there were large quantitative differences between genotypes in
277
both the leaf and the stem phenolics. This was proven by the PERMANOVA analysis, where the largest part of the
278
variation in the leaf phenolic profiles was explained by genotype (Table 3). High genotypic variation is seen in the PCA
279
plots also and in the high standard error values in the concentrations (Table 1; Table 2; Table S2; Figure S3; Figure S4).
280
In the stems, all the individual phenolic acids, phenolic glycosides and salicylates were detected in all of studied the
281
genotypes, while in the leaves only all the individual phenolic acid compounds were detected in all of the genotypes.
282
One individual leaf salicylate, tremulacin, and almost half of the total number of individual leaf and stem flavonoid
283
compounds, such as quercitrin in the leaves and quercetin 3-arabinoside in the stems, were detected in only some of the
284
genotypes. Condensed tannin concentration in the leaves of some of the genotypes was ten times higher than in the
285
others. In the PCA of leaf phenolic compounds, four genotypes stood out from the others quite clearly (Figure S3).
286
These distinctions were mainly explained by the high concentrations of (+)-catechin and condensed tannins in
287
genotypes 15 and 19, and tremulacin in genotypes 10 and 11.
288 289
Growth and Flowering. The height and diameter growth of the plants was steady over the years (Figure 3), and there
290
were no significant trends in the studied leaf parameters (leaf dry weight, leaf area and specific leaf area, SLA) with
291
increasing age (Figure 4). On the other hand, yearly weather conditions seemed to have an effect on leaf size; leaves
292
were largest and thickest during the warm summers 2010–2011 (mean temperatures during the months from June to
293
August 16.5 °C and 16.8 °C, respectively, compared to the mean for thirty years, 15.5 °C), and during a rainy summer
294
2012 (mean precipitation 301 mm from June to August, compared to the mean for thirty years, 75.5 mm). Females and
295
males did not differ in height and diameter growth, nor in any of the leaf size parameters (Figure 3, 5). S. myrsinifolia
296
started to flower at the age of three to four years, in 2009–2010. Number of catkins per shoot was higher in male
297
genotypes in 2009, while in 2010 there was no difference in flowering between sexes (significant year*sex interaction,
298
F (1, 663.5)=5.782 P<0.05; Figure 5).
299 300
Rust Damage. There was no significant effect of age or difference between females and males in the number of rust-
301
infected plants, nor in the severity of rust-infection in the leaves of S. myrsinifolia (Figure 6). There was, however,
302
some variation between years. Rust infections were severe in 2012, when the mean summer temperature was low (14.7
303
°C) and it rained frequently (301 mm) (Figure 6). On the other hand, rust infections were rare in 2010 and 2011, when
304
the mean summer temperature was high and it rained less, as mentioned above (156 mm and 196 mm, respectively)
305
(Figure 6). Neither the PERMANOVA (only 7.9% of explained variation) nor the PCA-analysis showed any strong
306
effects of the degree of rust-infection on the phenolic profile of the leaves in comparison to the effect of genotype
307
(Table 3; Figure S3).
308 309
DISCUSSION
310 311
This study gives a unique overview of the defensive phenolic profiles of leaves and stems of a Salicaceae species across
312
plant ontogenetic stages, and over a long-term study period (up to seven years). To our knowledge, this is the first time
313
that detailed phenolic profiles were analyzed from the same plant individuals and from the same microclimatic
314
environment for such a long period. As we hypothesized, age did not influence the concentrations of phenolic
315
compounds in the leaves or stems of S. myrsinifolia, which maintained constant defense levels over ontogeny after the
316
sapling phase and after reaching the reproductive phase. The concentration levels of different low molecular weight
317
phenolic groups and their relative proportions in the leaves and stems of S. myrsinifolia corresponded well with those
318
found in previous short-term studies of the same species in the field (Nybakken et al. 2012; Randriamanana et al.
319
2015a; Tegelberg et al. 2003). Yearly variation in the climatic conditions in the field may have reflected in changes in
320
leaf size, and may explain the detected slight variation in the concentrations of most of the studied compounds in the
321
leaves.
322
We detected only a few changes in stem phenolics and basically no changes in leaf phenolics with age
323
in S. myrsinifolia. The few significant changes that were detected with age in the concentrations of low molecular
324
weight phenolic compounds in the stems were directed towards lower amounts in older plants, contrary to what we
325
hypothesized. This may partly be a result of dilution (Koricheva 1999).
326
Salicylates, which represent a key group of the studied phenolics, have been shown to be largely
327
ineffective against specialized insect herbivores, such as the leaf beetle Phratora vitellineae (Sipura and Tahvanainen
328
2000). It is thus probable that the salicylates in S. myrsinifolia were primarily targeted at generalist insect herbivores
329
(Ruuhola et al. 2001; Tahvanainen et al. 1985; Volf et al. 2015) or small mammalian herbivores, such as hares (Lepus
330
ssp.) and voles (Microtus ssp.), which have been shown to be important herbivores of S. myrsinifolia (Heiska et al.
331
2008; Shaw et al. 2010; Tahvanainen et al. 1985). The constantly high levels of leaf phenolics in S. myrsinifolia are in
332
contrast to decreasing levels of phenolics in basal Salicaceae species. For example, salicylates are replaced by other
333
phenols, such as flavonoids and condensed tannins in the later ontogenetic phases of S. pentandra and P. tremula
334
(Donaldson et al. 2006, Julkunen-Tiitto and Virjamo 2017, Nissinen et al. 2017). These large tree species are less
335
exposed to herbivory by smaller mammals when mature, which may explain the decrease in salicylates with age. On the
336
other hand, the relatively smaller S. myrsinifolia remains exposed to mammalian herbivores even when mature, possibly
337
explaining its steady levels of phenolics. Indeed, larger Salix species have been reported to have lower concentrations of
338
phenolic compounds compared to the smaller sized S. daphnoides and S. purpurea (Förster et al. 2008, 2010;
339
Tahvanainen et al. 1985). Unfortunately, the phylogeny of the Salix species remains largely unresolved, and the
340
information of developmental changes in willow defenses is rather scarce (Lauron-Moreau et al. 2015; Liu et al. 2016;
341
Wu et al. 2015). We are thus not able to say whether the contrasting defensive strategies between S. myrsinifolia and the
342
basal Salicaceae species truly result from their different growth forms or from an evolutionary shift in defensive
343
strategies (Lauron-Moreau et al. 2015; Liu et al. 2016; Wu et al. 2015).
344
We hypothesized that males would be more growth-oriented, while females would invest more in
345
defensive compounds and reproduction. However, females and males of S. myrsinifolia differed little in the studied
346
parameters. Our results are in accordance with earlier field studies, but disagree with results from greenhouse studies
347
(Nybakken and Julkunen-Tiitto 2013; Nybakken et al. 2012; Randriamanana et al. 2014, 2015a). In greenhouses, plants
348
have optimal growing conditions, and thus the responses are not fully comparable to those found in the field. Several
349
concurrent stress factors (e.g. herbivory, plant deseases, competition by other plants, heat, heavy rain and strong wind)
350
in the field seem to level out possible sexual differences in general. Only one chlorogenic acid derivative was
351
significantly more abundant in the leaves, as was neochlorogenic acid in the stems of females. Chlorogenic acid
352
concentrations were also higher in females in field studies of one- to three-year-old S. myrsinifolia (Nybakken et al.
353
2012; Randriamanana et al. 2015a). Chlorogenic acids act as a deterrent against various leaf beetles and thrips (Leiss et
354
al. 2009; Matsuda and Senbo 1985). Chlorogenic acids may also serve as a precursor for the synthesis of flavonoids in
355
the phenylpropanoid pathway. In consequence, the higher concentrations of chlorogenic acids in females may indicate
356
that they rely more on inductive defenses than males do (Nybakken et al. 2012). Low number of significant sexual
357
differences detected may be also a consequence of the considerable differences in the concentrations of all the analyzed
358
compounds among the genotypes.
359
We found substantial genotypic differences in the concentrations of all analyzed phenolic compounds
360
in the leaves and stems of S. myrsinifolia. For example, tremulacin was found only in two genotypes. In addition, we
361
found large quantitative differences in the concentration of condensed tannins. The notable high genotypic variation in
362
phenolics among the Salicaceae species has been shown in several studies (e.g. Hakulinen and Julkunen-Tiitto 2000;
363
Paajanen et al. 2011; Randriamanana et al. 2014). An earlier study of S. myrsinifolia concluded that the genotypic
364
differences in phenolic concentrations were even greater than the differences between Salix species (Tegelberg et al.
365
2003). Genotypic variation allows plant species to survive in a larger range of environments and in changing conditions
366
(Liao et al. 2016). Furthermore, previous studies have demonstrated that closely related congeneric plants tend to
367
diverge in their defenses, which probably allows them to escape herbivory (Becerra 2007; Kursar et al. 2009; Salazar et
368
al. 2016; Volf et al. 2018). Similarly, intraspecific variation can reduce a pool of shared herbivores between S.
369
myrsinifolia genotypes.
370
As we hypothesized, age did not affect the severity of rust infections in S. myrsinifolia during the
371
seven-year period. However, there were substantial differences among years in the severity of rust infections. This is
372
probably a result of varying microclimate factors, such as humidity and temperature conditions. In general, for different
373
rust species, sufficient moisture is a prerequisite to thrive (Helfer 2014). In earlier studies of S. myrsinifolia and P.
374
tremula, rust severity was lower in rapid-growing genotypes and under elevated temperature (Heiska et al. 2007;
375
Nybakken et al. 2012; Randriamanana et al. 2015a, 2015b). Accordingly, we observed a clear relationship between
376
weather conditions and severity of rust infections. The leaves were heavily infected by rust in cold and rainy summers,
377
whereas during warmer and drier summers the infection rates were low. Many of the leaf phenolics have been found to
378
be inducible by different stress factors (Boege 2004; Coleman and Jones 1991; Karban and Myers 1989; Raupp and
379
Sadof 1991). The accumulation of both condensed tannins and salicin may have been induced by rust-damage in the
380
six-year old S. myrsinifolia plants (e.g. Hjältén et al. 2007), and the increase in total phenolics in the seven-year old
381
plants may suggest a delayed induction response to the severe rust attacks of the previous year. Severe damage may
382
have caused a regeneration process in the plants and thereby increased the synthesis of defensive compounds. For
383
example, Miranda et al. (2007) found a connection between rust-infection and the activity of genes encoding condensed
384
tannin synthesis in Populus trichocarpa x P. deltoides. On the other hand, high levels of salicin may also be a result of
385
the degradation of more complex salicylates, such as salicortin, which concentration decreased at the same time.
386 387
Conclusions. S. myrsinifolia has a high constitutive defense profile, which may have evolved as a consequence of being
388
constantly exposed to a high herbivore pressure. Concentrations of different phenolic compounds remained more or less
389
at a constant level over a seven-year period in both leaves and stems. The moderate yearly variation detected in
390
phenolic concentrations seemed to be highly affected by changing weather conditions as well as rust infestation levels.
391
The high recovered variability in polyphenol profiles among S. myrsinifolia genotypes makes this species an ideal
392
model for studying how genotypic variability in natural willow communities affect their chemical diversity and how
393
this is mediated to other trophic levels, such as rich communities of willow herbivores.
394 395
Acknowledgements–Our sincere thanks are owed to Sinikka Sorsa, Hannele Hakulinen, Riitta Pietarinen and Outi
396
Nousiainen for their help with laboratory analyses and Mervi Kupari, Teija Ruuhola, Anneli Salonen, Norul Sobuj,
397
Meeri Koivuniemi, Riia Hörkkä, Anu Ovaskainen and Henna-Riikka Leppänen for their help with field measurements,
398
leaf and stem sampling and in rust and herbivory damage assessment. We are also indebted to the reviewers for their
399
valuable and thorough comments on the manuscript. The English language of the article was checked by Rosemary
400
Mackenzie. This work was financed by the Academy of Finland (no. 267360).
401 402
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