Ecological Research (2001) 16, 803–808
NOTE AND COMMENT Nutrient compositions of acorns and horse chestnuts in relation to seed-hoarding Takuya Shimada*
Kansai Research Center, Forestry and Forest Products Research Institute, Momoyama, Kyoto 612-0855, Japan Nutritional compositions of acorns (Quercus serrata Thunb. and Quercus mongolica Fisch. var. grosseserrata Rehd. et Wils.) and horse chestnuts (Aesculus turbinata Blume) were analyzed. Major nutrients of acorns and horse chestnuts were carbohydrate, protein and fat. They proved to contain considerable amounts of tannins (7.28–11.72% dry mass-1) and saponins (6.20%), respectively. The nutrients and the secondary metabolites of seeds buried for either 1 month or 3 months were analyzed to evaluate effects of seed-caching by animals. Noticeable changes were not observed, which suggested that caching may not bring any advantages to the seed-hoarders with respect to nutrition. Key words: Aesculus (Hippocastanaceae); plant chemical defenses; Quercus (Fagaceae); saponins; tannins.
INTRODUCTION Acorns, seeds of genus Quercus (Fagaceae), and horse chestnuts, seeds of genus Aesculus (Hippocastanaceae), provide an abundant resource for wildlife. However, feeding on them brings potential costs to animals. Acorns have tannins, and horse chestnuts contain saponins as plant secondary metabolites (Kariyone & Tobinaga 1958; Ofcarcik & Burns 1971; Short 1976; Shimada 2001). Tannins, a diverse group of water-soluble phenolics, are thought to be defensive products against herbivory by reducing protein digestibility (Zucker 1983). Recent researches, however, suggested that tannins should be recognized not only as digestion inhibitors, but also as potential toxins causing direct detrimental effects to the gut epithelium, intrinsic nitrogen loss and disturbance of Na balance (Freeland & Janzen 1974; Zucker 1983; Robbins et al. 1987a, 1987b; Thomas et al. 1988; Hagerman & Robbins 1993; Dearing 1997a).
*Email:
[email protected] Received 25 December 2000. Accepted 23 May 2001.
Saponins, a group of steroid or triterpenoid glycosides, have effects of inhibiting digestion and hemolysis (Gershenzon & Croteau 1991). To clarify the nutrient composition is the first step to evaluate antinutritional effects of acorns and horse chestnuts on herbivores. However, reliable information about their nutrients has been poor. Seed predators, such as wood mice, squirrels and jays, cache seeds in the ground before feeding (see review: Vander Wall 1990). Animals may manipulate, via caching, the nutritional composition, particularly the levels of secondary metabolites (Roy & Bergeron 1990; Dearing 1997b). The objectives of this study are: (i) to clarify the nutrient compositions of acorns, Quercus serrata Thunb. and Quercus mongolica Fisch. var. grosseserrata Rehd. et Wils., and Japanese horse chestnuts, Aesculus turbinata Blume; and (ii) to evaluate the influences of caching on chemical constituents in these seeds, particularly the secondary metabolites, tannins and saponins. Quercus serrata is common in warm temperate forests, and Q. mongolica var. grosseserrata and A. turbinata commonly occur in cool temperate forests (Kitamura & Murata 1971). These three species are deciduous canopy trees. Acorns of Q. serrata and Q. mongolica var. grosseserrata are about 16–22 mm and
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20–30 mm in length, respectively (Kitamura & Murata 1971). Whereas Q. mongolica var. grosseserrata exhibits masting (Imada et al. 1990), seed production of Q. serrata is relatively constant (Fujii 1993). Wood mice (Apodemus species), voles (Clethrionomys and Eothenomys species), chipmunks (Tamias sibiricus Laxmann), squirrels (Sciurus species), and jays (Garrulus glandarius Temminck et Schlegel) are known to be major seed hoarders of these acorns (Miyaki & Kikuzawa 1988). Seeds of A. turbinata are large (30–40 mm in diameter) and chestnut like (Hoshizaki et al. 1999). Fluctuations in seed production of A. turbinata are small (Hoshizaki et al. 1997). Seeds of A. turbinata are hoarded mostly by Apodemus speciosus Temminck (Isaji & Sugita 1997; Hoshizaki et al. 1999). Nomenclatures were followed by Kitamura and Murata (1971) for plants, Abe et al. (1994) for mammals, and Wild Bird Society of Japan (1982) for birds.
METHODS Quercus serrata acorns from Kyotanabe in Kyoto prefecture (34°48¢N, 135°46¢E), Q. mongolica var. grosseserrata acorns from Miyama in Kyoto prefecture (35°16¢N, 135°44¢E), and Japanese horse chestnuts from Mikata in Hyogo prefecture (35°15¢N, 134°30¢E) were used for analyses. Acorns were collected in October 1999, and horse chestnuts were collected in September 1999. Mature and sound seeds (approx. 200 g), without deterioration and damage by insects, were prepared for the experiment. Half of the seeds prepared were buried in the research forest of Forestry and Forest Products Research Institute, Kansai Research Center, Kyoto (34°56¢N, 135°46¢E). The research forest is an evergreen forest in which Pasania edulis Makino (Fagaceae) and Quercus myrsinaefolia Blume (Fagaceae) are dominant. Acorns with their seed coat removed and horse chestnuts cut into halves were set at approximately 3 cm depth and covered with soil to simulate the situation that rodents eat a part of seeds and cache them (Isaji & Sugita 1997; T. Shimada, unpubl. obs., 1995). Seeds were buried on 28 October 1999 for two different periods: approximately 1 month, or 3 months. For the 1-month-buried sample, seeds were recovered
on 2 December 1999. For the 3-month-buried sample, seeds were recovered on 27 January 2000. Recovered seeds were washed lightly by distilled water and the surface was wiped up with filter paper. Soil particles penetrated into the plant tissue were not removed. I served the rest of the seeds as a ‘fresh’ sample. The seed coat of acorns was excluded but, for horse chestnuts, whole seeds were used due to difficulty of removing the seed coat from the kernel. Items measured were water content, crude protein, crude fat, crude fiber, crude ash, total phenolics, saponins (only for horse chestnuts), tannin astringency (protein-precipitating ability), and calories (only for fresh samples). For the 3month-buried sample, only water content and tannin astringency were determined. I entrusted the measurements of the following items to the Japan Food Research Laboratories; crude fat (diethyl ether extraction method), crude fiber (The Japan Society of Analytical Chemistry 1981), total phenolics (Folin-Denis method using 50% v/v MeOH as the extract and tannic acid as the standard; Folin & Denis 1912; Waterman & Mole 1994), saponins (n-BuOH extraction method, Kitagawa et al. 1984), and calories (bomb calorimetry). A part of the samples was roughly crushed and put in an electric dry oven set at 105°C for 24 h and water content was calculated. The rest was milled for further analyses. Crude protein was measured as nitrogen ¥ 6.25 by NC-analyzer (NC-800; Sumika Chemical Analysis Service Ltd, Osaka). A small amount of milled sample (approx. 5 g) was burned in the muffle furnace set at 550°C and the rest was regarded as crude ash. Nitrogen-free extract (NFE) is a residual fraction and expressed as the remainder of the other nutrients: water content, crude protein, crude fat, crude fiber, and crude ash. Carbohydrate is a major component of NFE. Total phenolics include both tannins and nontannic phenolics, which are not able to precipitate proteins (Zucker 1983). Thus, the radial diffusion method for the index of the tannin astringency was employed (i.e. the ability of precipitating proteins (Hagerman 1987)). Milled sample (approx. 100 mg) and 50% v/v MeOH (0.5 ml) was placed into a microtube and it was bathed for 30 min in an ultrasonic bath for extracting tannins. Wells were made in agarose plates containing 0.1% w/v
Aesculus turbinata
Quercus mongolica var.grosseserrata
805 *Tannin astringency is expressed as TAE (mg g-1 seed dry wt.). Fat, fiber, total phenolics and saponin were measured by Japan Food Research Laboratories. Saponin of Quercus acorns was not determined. Nutrient compositions and total phenolics of 3-month-buried samples were not determined. NFE, nitrogen-free extract; mo., month; mos. months.
26.5 57.2 33.4 85.7 100.5 100.8 Not detected Not detected Not detected – – – – – – 6.2 6.4 – 7.3 7.5 – 11.7 12.5 – 0.4 0.6 – 88.3 87.4 – 90.3 88.3 – 81.0 80.6 – 2.8 3.3 – 1.5 2.7 – 2.7 1.8 – 1.9 2.4 – 2.1 2.6 – 4.1 6.8 – 2.5 2.0 – 1.7 1.5 – 3.9 5.5 – Fresh 1 mo. buried 3 mos. buried Fresh 1 mo. buried 3 mos. buried Fresh 1 mo. buried 3 mos. buried Quercus serrata
4.5 4.9 – 4.4 4.9 – 8.2 5.4 –
Tannin astringency Saponin Tannin phenolics Total NFE Crude ash Crude fiber Crude fat Crude protein
Water content was approximately 50% for all the samples analyzed (Q. serrata fresh 43.1%, 1month-buried 50.5%, 3-month-buried 46.3%; Q. mongolica var. grosseserrata fresh 52.3%, 1-monthburied 54.4%, 3-month-buried 56.6%; A. turbinata fresh 51.6%, 1-month-buried 54.3%, 3-month-buried 49.2%). Calorie per g dry mass of the seeds was approximately the same value among the three species (Q. serrata fresh 17.6 kJ g-1; Q. mongolica var. grosseserrata fresh 18.0 kJ g-1; A. turbinata fresh 19.7 kJ g-1). Table 1 gives the results of nutrient analyses of acorns and horse chestnuts. Values are expressed as percent per g dry weight of the seeds. Nitrogen free extract was a major component of nutrients in all species (80–90% of the dry mass). Horse chestnuts had higher protein, fat and fiber content than the two species of acorns. The content of crude ash was almost the same among the three species. The composition of nutrients was quite similar in the two acorn species, but Q. serrata had slightly higher content of fat and ash than Q. mongolica var. grosseserrata. Acorns and horse chestnuts both have tannins and saponins as secondary metabolites (Table 1). The acorns of the two Quercus assayed quite high in total phenolics (Q. serrata fresh 7.3%; Q. mongolica var. grosseserrata fresh 11.7%). Whereas the content of total phenolics in Q. mongolica var. grosseserrata was 1.6 times higher than that of Q. serrata, the tannin astringency in Q. mongolica var. grosseserrata was about three times more intense than that
Treatment
RESULTS AND DISCUSSION
Species
bovine serum albumin with 4-mm punch, and an 8-µl aliquot of extract was applied to each well. After 120 h, the diameter of the ring that developed around the well was measured. The diameter squared is proportional to the amount of tannins added to the well. The standard curve was prepared using tannic acid and the value of the tannin astringency was expressed as mg tannic acid equivalent (TAE) per g dry mass of the seed. All chemicals were of either analytical grade or the highest purity grade available. Tannic acid was obtained from Dainippon Pharmaceutical Co. Ltd, Osaka.
Table 1 Nutritional constituents and tannin activity of seeds of two Quercus and one Aesculus. Each value of nutrient constituents is shown as percent content per dry weight*
Nutrient composition of seeds
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of Q. serrata (tannin astringency, Q. serrata fresh 26.5 TAE mg g-1; Q. mongolica var. grosseserrata fresh 85.7 TAE mg g-1). Fresh horse chestnuts contained a large amount of saponins and a small amount of phenolics, but did not exhibit tannin astringency. Tannins in acorns and saponins in horse chestnuts are likely to cause some antinutritional effects on seed predators. Further, Q. mongolica var. grosseserrata acorns, which have much higher tannin astringency, are supposed to be more deleterious than Q. serrata acorns. The results for Q. serrata by Shimada (2001) and for Q. serrata and Q. mongolica var. grosseserrata by Matsuyama (1982) agree with this study in spite of the difference of the source of acorns. The nutrient composition of acorns in North America has been well studied (Ofcarcik & Burns 1971; Short 1976), and the composition is different from that of Japanese Quercus studied here. Quercus species in North America are divided into two subgenera, Quercus and Erythrobalanus, which are called white and black oaks, respectively. According to Ofcarcik and Burns (1971), acorns of white oaks contain less tannin (0.6–2.1%) and a considerable amount of fat (4.6–11.5%), whereas those of black oaks are characterized by higher tannin (5.7–8.8%) and fat content (11.1–30.0%). Effects of burying on the nutrient composition of acorns and horse chestnuts were ambiguous (Table 1). Precipitation in these periods was 52.5 mm for the 1-month-buried sample and 121.5 mm for the 3-month-buried sample (Kansai Research Center FFPRI 1999; Kansai Research Center FFPRI, unpubl. data, 2000). Although they were slightly below the average for the preceding 5 years (67.3 mm and 167.4 mm, respectively; Kansai Research Center FFPRI 1994, 1995, 1996, 1997, 1998), the precipitations in the experimental periods were in the top three among these 6 years. Thus, it can be an average year in terms of precipitation, and precipitation may not be associated with these results. After 1-month-burying, some protein and ash was lost in horse chestnuts, whereas fat and fiber increased relatively. However, regarding acorns, a slight decrease of fat and a relative increase of protein, fiber and ash were observed. Total phenolics and saponin slightly augmented after 1month-burying. One- and 3-month-buried acorns exhibited higher tannin astringency than fresh
acorns. In Q. serrata the astringency of 3-monthburied acorns was intermediate between fresh and 1-month-buried samples. Contrary to this, the astringency of 3-month-buried acorns in Q. mongolica var. grosseserrata was almost equal to 1-month-buried samples. Dearing (1997b) found that levels of secondary metabolites in leaves cached by North American pikas had decreased with time to levels readily consumed by pikas and hypothesized that foodhoarding herbivores could manipulate the levels of these chemicals via caching. Roy and Bergeron (1990) also found that conifer branches, which were cut off and laid on snow by voles for several days before eating, lost tannins to similar levels to those found in their preferred foods. The results of this study do not support the above hypothesis. Similar results to this study have been reported using acorns in North America, which has suggested that caching has no effect on tannin levels in acorns (Dixon et al. 1997; Koenig & Faeth 1998). Although phenolics and saponins are water soluble (Kariyone & Tobinaga 1958; Zucker 1983), these chemicals in acorns and horse chestnuts may be more difficult to leach out than those in leaves or branches. This is probably because of the physical properties of acorns and horse chestnuts, which have a smaller ratio of surface area to volume.
ACKNOWLEDGEMENTS I am grateful to S. Taniguchi and M. Kashiwagi for providing horse chestnuts and acorn samples, S. Kaneko, H. Furusawa, K. Nambu and Y. Takahata for their advice in laboratory works, Y. Segawa and M. Kawashima for their assistance in nutrient analyses, and T. Saitoh and K. Hoshizaki for their constructive discussions.
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