Vegetation Science 37 : 101-107, 2020 Deer grazing on a plant with chemical defence 101 Short communication Severe grazing pressure on an unpalatable plant, Primula 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 species 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 plants 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.