Vegetation Science 37 : 101-107, 2020 Deer grazing on a with chemical defence 101 Short communication

Severe grazing pressure on an unpalatable plant, japonica, and its potential chemical compound for grazing defence in a long- term deer grazing habitat

Ryo O. SUZUKI1*, Yuri MAESAKO2 and 1Faculty of Science, University of the Ryukyus 2Graduate School of Human Environment, Osaka Sangyo University 3 Shigeru MATSUYAMA 3Graduate School of Life and Environmental Sciences, University of Tsukuba

We monitored the deer grazing pressure on a population of an unpalatable perennial, Primula japonica in a long-term grazed habitat by sika deer, using an enclosure treatment with cages for two years. The two-year census showed that all unenclosed individuals of this lost their aboveground parts due to grazing, where- as all enclosed individuals survived and most of them elongated inflorescences at the end of the census period. Although inflorescences extended outside cages, no grazing on the reproductive organs was observed. We also quantified the main chemical compound of the species using the gas chromatography and evaluated variations in the concentration of the compound among regions (a high deer-density site and a low deer-density site) and among organs (leaves and inflorescences), which identified flavone as the main compound and detected a higher flavone concentration in flowers than in leaves but little difference among regions. Although the results showed a possibility that flavone can contribute to protecting reproductive organs from deer grazing, we could not demonstrate clear evidence that flavone has toxic effects on deer. Future studies need to evaluate whether deer also graze other unpalatable in high-deer density regions and to verify mechanisms on how sika deer can overcome the chemical defence of unpalatable plants.

Key words: chemical defence, deer grazing, flavone, unpalatable plant

(Torii & Tatsuzawa 2009). It is reported that under ex- INTRODUCTION ■ tremely high densities of deers (called as deer overabun- Plant-animal interaction is one of the most important dance, Côté et al. 2004), even unpalatable plants can also topics in ecological and evolutionary studies (McCall & become subject to deer grazing (Kawahara 1992). Actual- Fordyce 2010). Many plant species evolved defensive ly, deer grazing pressure to unpalatable species, such as strategies to avoid from grazing pressure of herbivorous an- Maesa japonica (Maesako 2001) and Urtica thunbergiana imals, such as chemical defence with toxic materials and (Kato et al. 2008), are now observed in this region. There- physical defence with spines (these species with defensive fore, we expected that P. japonica plants would also begin strategies are called as unpalatable plants). Previous stud- to suffer from deer grazing. In 2001, we had observed a ies found that unpalatable plants have shown local adaption large number of reproductive plants of P. japonica at a site that further strengthens their defence strategy under severe of this region (Fig. 1a). In this study, we resurveyed the P. grazed-habiats (Kato et al. 2008; Suzuki 2008; Pardo et al. japonica population to check the grazing damage of this 2018). population. We report the deer grazing pressure on a population of Because the toxic materials of P. japonica are unknown, Primula japonica, in the Kasuga-Yama forest of Nara park we also examined to identify toxic compounds by chemical region. This species is known as an unpalatable plant with analysis and evaluate variations in the concentration of a chemical defence strategy (Takatsuki 1989). Because chemical compounds among regions (a high deer-density deer population density is extremely high in the region, site and a low deer-density site) and organs (leaves and Nara deer are exposed to severe food shortage, and many of flowers). We predicted that the concentration of toxic them would not be able to survive in a truly wild situation compounds in P. japonica was higher in reproductive or-

*Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan E-mail: [email protected] The Society of Vegetation Science ♦ Received March 19, 2020 ╱ Accepted September 11, 2020 102 Vegetation Science Vol. 37, No. 2, 2020

Fig. 1 A Primula japonica population in May 2001 (a), decreased in May 2011 (b), enclosed plants (c) and unenclosed plants (d) in May 2013. Deer grazing was observed for unenclosed leaves (d) but not on flowers exposed from cages (c). Circles in (b) show the position of P. Japonica individuals. Plant in (d) was newly emerged in May 2013. gans than leaves, based on the optimal defence theory pre- in this region for the years 1980-2013 (Japan Meteorologi- dicting higher allocation of chemical defences to higher cal Agency, https://www.data.jma.go.jp/obd/stats/etrn/in cost tissues (McCall and Fordyce 2010). We also predict- dex.php, accessed on 20 August, 2019). Sika deer (Cervus ed higher concentration of toxic compounds in high deer- nippon Temminck) populations have been distributed in density habitats than in low deer-density habitats due to lo- this forest for more than 1200 years, and the current popu- cal adaptation strengthening chemical defence. lation density (individuals / km2) of deer has reached 15.3 in March and 22.8 in November in the Kasuga-Yama forest (Torii et al. 2007). On 21 Sep. 2011, the 5 × 7 m plot was MATERIALS AND METHODS ■ established in a canopy gap in the area that Acer rufinerve Field population census dominated, where over 30 reproductive plants of P. japoni- Primula japonica is a perennial herb that is distributed ca have been observed in 2001 (Fig. 1a). All established widely throughout Japan and is common to swamps in for- individuals of P. japonica in the plot were tagged. On 23 ests and mountains (Satake et al. 1981). The population Apr. 2012, six individuals newly emerged were also tagged census of P. japonica was conducted in a warm-temperate (21 individuals in total), and five individuals randomly se- evergreen forest in Kasuga-Yama (34°41’N, 135°50’E) at lected were enclosed by cages of 30 cubic cm to prevent the eastern edge of Nara Prefecture, western Japan. Annu- deer grazing. The fate and feeding marks of each tagged al mean precipitation is 1015 mm, and monthly mean tem- individual were monitored on 3 Oct. 2012, and 24 May peratures range from 3.9°C in January to 26.9°C in August 2013. Deer grazing on a plant with chemical defence 103

Chemical analysis TSS2000 software (Shrader Analytical and Consulting Lab- Samples were collected from two sites; Kasuga-Yama oratories, Inc.). Identifications of the compounds were (34°41’02”N, 135°51’19”E) and Sakurai (34°27’53”N, tentatively done by library search with NIST mass spectral 135°45’14”E) in the Kinki area, west Japan (hereafter re- library (ver. 1.6), then confirmed by comparisons of mass ferred to as the Kasuga and Sakurai sites). The Kasuga spectra and retention times with those of authentic chemi- site was near to the population census plot in Kasuga-Yama cals. described above. The Sakurai site was located 25 km south of the Kasuga site. Although we have no clear evi- Statistical analyses dence of the exact density of deer at the Sakurai site, our Data analysis was conducted using R ver. 3.3.3. (R Core field observation found no clear traces showing deer graz- Team 2017). Kaplan-Meier survival curves of aboveg- ing at the Sakurai site. Based on our field observation, we round parts were compared between enclosed individuals assumed that deer density is lower at Sakurai site than the and unenclosed plants using a log-rank test by the survdiff Kasuga site. Three or four reproductive plants were col- function from the Survival library (Therneau 2015). In lected in July 2014 and May 2015 at the Kasuga site and in perennial plants, the disappearance of aboveground parts May 2015 at the Sakurai site. due to deer grazing cannot be definitely considered as indi- Each sample was divided into leaves, flowers, and inflo- vidual death, because their underground part may be alive. rescence. Samples in 2014 had no flowers due to the end Therefore, this study compared whether their aboveground of the reproductive period. Each organ was weighed and parts disappeared or remained at the end of the census peri- extracted with dichloromethane for 24 h. The extracts od. The concentration of the main chemical was calculat- were dried over anhydrous sodium sulfate and concentrated ed as the total amount of the main chemical per the fresh in vacuo. The residues were weighed and subjected to weight of organ extracted (leaves or flowers). The chemi- thin-layer chromatography (TLC), gas chromatography cal concentration in fresh leaves was compared among sites (GC) and GC coupled with mass spectrometry (GC-MS). in 2014-2015, and that in flowers was compared between Normal phase TLC analyses were conducted using a silica the Kasuga and Sakurai sites in 2015. After combining gel coated glass plates (Merck, Silica gel 60, F254, 5 cm × data of different sites and years, differences in the chemical 20 cm) with two different solvent systems (Solvent A: 50% concentration between fresh leaves and flowers were also diethyl ether in hexane, Solvent B: 10% methanol in chlo- tested. All comparisons were performed using Wilcoxon roform). Reversed-phase TLC was performed on What- rank-sum tests by the wilcox.test function in R. man KC-18F (5 × 20 cm) using 30%H2O in methanol as a developing solvent. Visualizations of the compounds RESULTS were done by exposing the plates under UV lights (254 nm, ■ 365 nm) followed by exposure to iodine vapor. For GC At the end of the census period (24 May 2013), all unen- analyses, a gas chromatograph (HP6890, Agilent Technolo- closed individuals lost their aboveground parts due to deer gies) equipped with a fused silica capillary column (007- grazing, whereas aboveground parts of all enclosed individ- FFAP, Quadrex, 25 m × 0.25 mm, 0.25 µm film thickness) uals remained and 4 of 5 enclosed individuals elongated in- was operated with an isothermal oven temperature of florescences (Fig. 1c). Although inflorescences extended 230°C. Temperatures of both injector and flame ionization outside the cages, no grazing on the reproductive organs detector were set at 250ºC. Helium was used as a carrier was observed. Survival analysis showed a significant dif- gas at 1 mL/min in the constant flow mode. Samples were ference in survival curves of aboveground parts between injected in the split mode (100: 1) and the chromatograms enclosed and unenclosed individuals (Fig. 2). were analyzed with Chemstation software (Agilent, ver. The extracts from leaves and flowers of P. japonica A10.01). GC-MS analyses were conducted using a gas showed a major UV spot at Rf = 0.12 (solvent A) and at Rf chromatograph (HP6890N, Agilent Technologies) coupled = 0.75 (solvent B) by silica gel TLC. The same spot ap- with a mass spectrometer (JEOL MS-600H). Samples peared at Rf = 0.13 by reversed-phase TLC. The mass were injected at 280°C in the split mode (100: 1) into a chromatograms of the extracts from flowers showed a sin- DB-5MS column (25 m × 0.25 mm, 0.25 µm film thick- gle peak at 20.72 min which appeared as a major peak con- ness, Agilent Technologies), separated under an oven tem- sisting of 61.1 - 86.0% of the total peak area in the extracts perature program of 50°C (1 min held) to 320°C at a rate of from leaves (Fig. 3). Mass spectrum of the major peak 10°C/min and held for 12 min. Carrier gas and the flow was suggested as flavone (2-phenylchromen-4-one) by rate was the same as GC analyses. Mass spectra were ob- mass spectral library search, which was confirmed by com- tained by electron ionization at 70 eV and analyzed by parisons of mass spectrum and retention time with those of 104 Vegetation Science Vol. 37, No. 2, 2020

■ DISCUSSION This study found that P. japonica individuals suffered from severe deer grazing in 2011-2013, and the loss rate of aboveground parts of unenclosed individuals was extremely high in the area. In 2015-2016, we re-surveyed the study population and two new populations of P. japonica in the Kasuga-Yama forest and found that deer grazing on P. ja- ponica was continuously observed and the grazing intensity varied among seasons, years, and populations (Maesako et al. 2018). The re-survey also observed an undesirable consequence that all enclosed individuals of P. japonica disappeared due to competition by grass and fern species established within cages. Considering those situations of recent grazing pressure and interspecific competition, both threats likely enhance the local extinction of the P. japonica Fig. 2 Kaplan-Meier survival curves of aboveground parts of enclosed plants (solid line) and unenclosed plants (doted line) population in the Kasuga-Yama forest. Our observation during a monitoring period from 21th Sep. 2011 to 24th May suggests that the P. japonica population should be main- 2013. tained under moderate grazing pressure. It is the first evidence identifying flavone from this spe- commercially obtained flavone (Fig. 4). In addition, Rf cies, whereas flavone was reported in Dionysia and Primula values of the authentic flavone (0.11: solvent A, 0.75: sol- species of (Valant-Vetschera et al. 2010). vent B) in TLC analyses coincided well with those of the Flavone in plants often has functions of flower pigments, major spot in the extracts. With all these data, it was con- such as anthocyanin, and freezing tolerance (Iwashina cluded that flavone was the main compound extracted from 2000; Isshiki et al. 2014). Our result showed that the fla- leaves and flowers of P. japonica. vone concentration in flowers was 4.6 times higher than Quantifications of the flavone in the extracts by GC -re that in leaves, suggesting that flavone acted as flower pig- vealed that there were no significant differences in the fla- ments. In contrast, flavone is known as one of the plant vone concentration in leaves among the Kasuga site in allelochemicals to exhibit biological activities on herbivo- 2014, 2015 and the Sakurai site in 2015. Moreover, there rous insects, such as increased mortality and growth inhibi- was no difference in the flavone concentration in flowers tion (Mahoney et al. 1989; Dowd & Shen 1990). Howev- between the Kasuga and the Sakurai sites in 2015. When er, previous studies found no evidence of the toxicity in combining data of different sites and years, the flavone con- flavone to mammals. centration in flowers (3.0 ± 0.8 mg g-1 fresh weight) was We propose a hypothesis that flavone is a potential al- significantly higher than that in leaves (0.6 ± 0.1 mg g-1 lelochemical relating to prevent deer grazing on the repro- fresh weight) (p = 0.0005, Wilcoxon rank-sum test). ductive organs. One circumstantial evidence supporting this hypothesis is that deer grazing was only observed on

Fig. 3 A typical mass chromatogram of leaf extract from Primula japonica. Deer grazing on a plant with chemical defence 105

Fig. 4 Mass spectrum of the major peak at 20.72 min (a) and that of authentic flavone (b).

leaves but not on flowers and inflorescences, whereas inflo- rescences extended outside the cages. This result suggests that there was a higher concentration of toxic chemicals in flowers than leaves. Because the large erect inflorescences growing up over 90 cm length in maximum are costly for P. japonica, the higher flavone concentration in reproductive organs may avoid from grazing damages. However, our circumstantial evidence is not sufficient, because there is a possibility that the reproductive organs were grazed after the end of the survey (24 May 2013). A previous study found higher concentration levels of toxic components in populations with stronger herbivory pressure and longer coevolutionary history with mammal herbivores (Pardo et al. 2018). On the other hand, our re- Fig. 5 Flavone concentration in leaves and flowers (mg g-1, sult showed no evidence that plants in a severe deer-grazed mean±SD) in the Kasuga site with high deer density and in habitat (the Kasuga site) possessed a higher concentration the Sakurai site with low deer densities in 2014-2015. Dif- of flavone than plants in a weak deer-grazed habitat (the ferent letters indicate significant differences among sites (P < Sakurai site). In conclusion, those results indicated no 0.05, Pairwise t-tests). clear evidence that flavone has toxic effects on deer. As mentioned above, flavone is more likely to be simply one 106 Vegetation Science Vol. 37, No. 2, 2020 of flower pigments. Further studies need to identify ef- Dowd, P.F. & Shen, S.K. 1990. The contribution of synbiotic fects of flavone on deer and examine why flowers of P. ja- yeast to toxin resistance of the cigarette beetle (Lasioderma ser- ricorne). Entomologia Experimentalis et Applicata, : 241- ponica were not grazed by deer in the Kasuga site. 56 248. The degradation of natural vegetation due to deer grazing Hashimoto, Y. & Fujiki, D. 2014. List of food plants and unpalat- has become one of the most serious environmental prob- able plants of sika deer (Cervus nippon) in Japan. Humans and lems in Japan (Takatsuki 2009). Increased deer pressure Nature, 160: 133-160 (in Japanese). causes change in species composition and diversity of plant Isshiki, R., Galis, I. & Tanakamaru, S. 2014. Farinose flavonoids are associated with high freezing tolerance in fairy primrose communities (Ohashi et al. 2007), and eventually can result (Primula malacoides) plants. Journal of Integrative Plant Biol- in irreversible degradation under excessive deer grazing ogy, 56: 181-188. (Sakaguchi et al. 2012). Unpalatable species also have Iwashina, T. 2000. The structure and distribution of the flavo- positive effects to maintaining plant diversity in grazed noids in plants. Journal of Plant Research, 113: 287-299. habitats (Callaway et al. 2000, 2005; Smit et al. 2006), by Kato, T., Ishida, K. & Sato, H. 2008. The evolution of nettle re- protecting other plants from deer grazing (Bossuyt et al. sistance to heavy deer browsing. Ecological Research, 23: 339-345. 2005; Osem et al. 2007; Suzuki & Suzuki 2011). As Kawahara, H. 1992. The effect of Nozaki-jima Sika deer forage shown in this study, the situation in which unpalatable habits on the island flora. Bulletin of Nagasaki Institute of Ap- plants are grazed by deer may further accelerate vegetative plied Science, 33: 137-144 (in Japanese). degradation by the loss of indirect positive effects of unpal- Maesako, Y. 2001. Change in plant palatability of deer grazing in atable plants. Nara Park and Kasugayama Reserved Forest, Nara, Japan. Nara botany, 23: 21-25 (in Japanese). In Japan archipelago, at least 135 plant species are identi- Maesako, Y., Suzuki, R.O., Hirashiba, K. & Nishiura, D. 2018. fied as unpalatable for deer (Hashimoto & Fujiki 2014). A Grazing pressure of sika deer (Cervus nippon) on an unpalatable comprehensive study is needed to evaluate whether deer are species, Primula japonica, in a warm-temperate evergreen for- beginning to graze these unpalatable plants and these plants est, Japan. Bulletin of Kansai Organization for Nature Conser- vation, 40: 23-33 (in Japanese). are facing local extinction in high-deer density regions. Mahoney, N.E., Roitman, J.N. & Chan, B.C. 1989. Structure-ac- Moreover, future studies need to verify mechanisms on how tivity relationship of flavones as growth inhibitors of the navel sika deer can overcome the chemical defence of unpalatable orangeworm. Journal of Chemical Ecology, 15: 285-290. plants. McCall, A.C. & Fordyce, J.A. 2010. 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