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Received: 12 January 2017    Revised: 28 April 2017    Accepted: 3 May 2017 DOI: 10.1002/ece3.3155

ORIGINAL RESEARCH

Seasonal changes in leaf chemistry and leaf selection of the Japanese giant flying squirrel upon two tree species Mutsumi Ito1

 | Noriko Tamura2 | Fumio Hayashi1

1 Department of Biology, Tokyo Metropolitan University, Hachioji, Tokyo, Japan

Abstract

2 Tama Forest Science Garden, Forestry and Forest Products Research Institute, Hachioji, Tokyo, Japan

Tree leaves are important food sources for arboreal herbivores, such as primates, ro-

Correspondence Mutsumi Ito and Fumio Hayashi, Department of Biology, Tokyo Metropolitan University, Hachioji, Tokyo, Japan. Emails: [email protected] and [email protected]

sugars, fibers, proteins, and toxins. However, the effects of the microscale distribution

Funding information Japan Science Society, Grant/Award Number: 28-­513 and Japan Society for the Promotion of Science, Grant/Award Number: 17J04373.

dents, and marsupials. These animals do not eat leaves randomly in habitats with many tree species but rather choose based on the chemical components of leaves, such as of these chemicals within each leaf have not been examined for these animals. The giant flying squirrels Petaurista leucogenys are entirely arboreal, nocturnal herbivores, usually feeding on leaves and dropping leaf debris on the ground after partially consuming them. Therefore, we could easily assess which species of trees and which parts of the individual leaves they preferred to eat. We also examined microscale distributions of phenolics, sugar, and water within individual leaves. Of the two dominant food tree species, the deciduous Quercus acutissima was preferred over the evergreen Q. sessilifolia. The latter tree is only used during winter to early spring when the former had no leaves. Our chemical analyses revealed that Q. acutissima contained much more glucose than Q. sessilifolia in all seasons. Three types of leaf debris, eaten apically, basally, or centrally with a hole, were found. In Q. sessilifolia, which had low phenolic concentrations, apical eating was most common, whereas central eating was rare. In Q. acutissima, which had high phenolics, basal or central eating was common. Central feeding may be caused by avoiding the periphery because of a higher phenolic concentration in the leaf margin. Thus, microscale distributions of phenolics within individual leaves affect which parts P. leucogenys eats, whereas leaf sugar concentration is an important factor affecting which species of leaves they eat. KEYWORDS

folivory, food selection, glucose content, leaf-folding, total phenolic content

1 | INTRODUCTION

(e.g., cellulose, lignin, and hemicellulose), which are poor energy sources,

Herbivorous mammals are forced to make optimal choices regarding their

and alkaloids) and digestion inhibitors (condensed tannins) (Farmer, 2014;

daily diets by selecting plants with higher nutritional value and by avoiding

Glander, 1982; Harborne, 1991). Food selection by herbivorous mammals

those with possible toxins to maintain and develop their bodies (Farmer,

based on the abundance of valuable nutrients and plant defensive chem-

2014; Glander, 1982). The main nutritional resources from plants are sug-

icals has been well documented and, in most cases, they select food ma-

ars, proteins, and minerals; some species have higher concentrations of

terials with substantial amounts of preferable nutrients (Jensen, Wallis, &

these than others (Farmer, 2014). However, plants usually include fibers

Foley, 2015; Kar-­Gupta & Kumar, 1994; Matsuda, Tuuga, Bernard, Sugau,

and also develop secondary metabolites, such as toxins (e.g., phenolics

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 5766  |  www.ecolevol.org 

Ecology and Evolution. 2017;7:5766–5773.

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into absorbable nutrients in P. alborufus lena of Taiwan (Lu et al., 2012). P. leucogenys usually eats leaves from a variety of tree species in the seasonal environment of Japan and chooses different food items (Ando, Shiraishi, & Uchida, 1985; Baba et al., 1982; Kawamichi, 1997, 2010). However, the effect of leaf chemicals on food choice has not been examined. We predict that P. leucogenys will prefer leaves with higher concentrations of soluble sugars and will avoid leaves with higher concentrations of phenolics. To examine this hypothesis, the leaf chemicals and nutritional conditions of two dominant Quercus tree species, deciduous Quercus acutissima and evergreen Q. sessilifolia, were assessed seasonally in relation to food choice by P. leucogenys.

2 | MATERIALS AND METHODS F I G U R E   1   Three types of leaf debris eaten by the Japanese giant flying squirrel (Type A, apically eaten; Type B, basally eaten; Type C, only centrally eaten). The total length (Lt) and width (Lw) of intact leaves are measured, as well as the remaining length for basally (La) and apically (Lb) eaten leaves, and the maximum width of the centrally eaten circle (Ld) of leaf debris. All leaves shown are the evergreen Quercus sessilifolia

2.1 | Study site and sample collection The single census route (2 km long, 5 m wide) was set in a 50-­ha wooded area in the Tama Forest Science Garden at Todori, Hachioji, Tokyo, Japan (35°38′50.74″N, 139°16′38.15″E). The vegetation was mixed temperate broadleaf and coniferous trees (Tamura, 2004). The climate is characterized by four seasons: spring (March to May), summer (June to August), autumn (September to November), and winter (December

& Hanya, 2013; Remis, 2002; Remis & Kerr, 2002; Willig & Lacher, 1991).

to February). Normal value average temperatures from 1981 to 2010 at

Many mammals are considered to have four primary taste modalities

Hachioji meteorological station are 3.2°C in January, 4.1°C in February,

(sweet, salty, bitter, and sour), with umami, a savory taste associated with

7.5°C in March, 13.1°C in April, 17.6°C in May, 20.9°C in June, 24.7°C

specific amino acids, being a fifth modality, as in humans (Lemon, 2015;

in July, 26.1°C in August, 22.2°C in September, 16.4°C in October,

Yarmolinsky, Zuker, & Ryba, 2009). Sweet and umami promote feeding

10.7°C in November, and 5.7°C in December (Japan Meteorological

behaviors, whereas bitter and sour tastes reduce food intake, and the per-

Agency, 2017). Monthly rainfalls are 48.3 mm in January, 49.4 mm in

ceptions of sweetness may contribute to preferences for fruits, flowers,

February, 103.4 mm in March, 118.2 mm in April, 121.5 mm in May,

and other foods high in soluble sugars (Yarmolinsky et al., 2009). Clear

167.9 mm in June, 176.0 mm in July, 242.2 mm in August, 256.7 mm in

preferences for sweetness and/or soluble sugar have been documented

September, 187.7 mm in October, 88.8 mm in November, and 45.2 mm

in animals ranging from relatively small mammals, such as frugivorous

in December (Japan Meteorological Agency, 2017).

bats (Ayala-­Berdon et al., 2011; Law, 1993), to large mammals, such as

The morning census was conducted usually from 9 a.m. along this

roe deer and primates (Remis, 2002; Remis & Kerr, 2002; Tixier et al.,

route, walking slowly for about 1.5 hr. It was conducted one to 5 days

1997). In contrast, a significant number of plant metabolites are bitter or

per month from May 2013 to November 2015, resulting a total of 87

otherwise unpleasant in taste (Harborne, 1991). The taste of bitterness

censuses. Along the route, we collected and counted all leaf debris

may predict the deterrent effects of toxins and digestibility-­reducing tan-

of Q. acutissima and Q. sessilifolia that had been eaten by P. leucog-

nin compounds in fruits and leaves (Critchtey & Rolls, 1996) and may be

enys. Direct observations of feeding are necessary to demonstrate

a principal cause of food rejection (Remis, Dierenfeld, Mowry, & Carroll,

their foods (e.g., Kawamichi, 1997; Koli, Bhatnagar, & Sharma, 2013).

2001; Takemoto, 2003). Therefore, mammalian herbivores often show

Although we did not observe directly their feeding at night, it has been

behavioral avoidance and/or tolerance of plant secondary metabolites,

well documented for P. leucogenys to remain the characteristic leaf

such as high concentrations of tannins and related polyphenolics (Glander,

debris when they eat tree leaves (Kawamichi, 1984a, 2010; Okazaki,

1982; Iason & Villalba, 2006; Sorensen, McLister, & Dearing, 2005).

2004; Shigeta & Okazaki, 2001; Shigeta, Shigeta, & Tamura, 2010). In

Tree leaves are important foods for arboreal herbivores, such as

this study therefore we used the number of such characteristic leaf

primates, rodents, and marsupials (Farmer, 2014). The Japanese giant

debris as the feeding intensity of P. leucogenys upon the two tree spe-

flying squirrel (Petaurista leucogenys Temminck, 1827) is an exclusively

cies. P. leucogenys was the only mammal eating tree leaves around the

arboreal, nocturnal, and tree leaf-­eating mammal (Ohdachi, Ishibashi,

census route (Mikuriya, 1993). Collected leaves were classified into

Iwasa, Fukui, & Saitoh, 2015). Its home range size is 0.4–5.2 ha, usu-

three types: eaten at the apical part (Type A), eaten at the basal part

ally larger in males, with considerable overlaps between the sexes and

(Type B), and eaten at the central part (Type C) (Figure 1). The remain-

between males (Baba, Doi, & Ono, 1982; Kawamichi, 1984b). Mature

ing lengths of basally and apically eaten leaves (La and Lb, respectively,

P. leucogenys reaches weights of up to 1.3 kg (Ohdachi et al., 2015).

in Figure 1) were measured with a slide calliper to the nearest 0.1 mm.

Such large bodies may be maintained by nutrition from specialized

In Type C leaves, the maximum width of the hole was also measured

cecal microbiota, which are known to convert diverse plant materials

(Ld in Figure 1). We collected several intact leaves during the censuses

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F I G U R E   2   (a) The mean (± SD) number of the leaf debris found in each month (n, the number of censuses per month). When no leaf debris was observed, it was treated as 0.1 (−1 on log10 axis). (b) The proportions of the three types of leaf debris, Types A (white), B (shaded), and C (black), in each month (n, total leaf debris per month). In both (a) and (b), the upper panel shows the leaves of deciduous Quercus acutissima, and the lower panel shows those of evergreen Q. sessilifolia and measured the length excluding the petiole (Lt) and the width at the

10 min and centrifuged at 2,500 g for 10 min. The supernatant was

widest part (Lw) as controls (Figure 1).

stored in a 15-­ml tube. This procedure was repeated three times, and the entire 3-­ml aqueous acetone extract was fully evaporated under

2.2 | Total phenolic content

low pressure at 40°C. The residue was dissolved in 1 ml distilled water. After being diluted 60 times, 0.3 ml of the solution was mixed

Fresh leaves of Q. acutissima and Q. sessilifolia were collected from

with 0.3 ml of 2N Folin reagent (Folin–Ciocalteau’s phenol reagent) in

the branches at the study site on 15 and 17 occasions, respectively,

a 2.0-­ml tube, allowed to stand for 5 min, and then mixed with 0.6 ml

from May 2013 to October 2015. These leaves were put in an ice

20% Na2CO3. After a 10-­min incubation at room temperature, the

box for 1 hr during transport and then stored at −30°C in a laboratory

mixture was centrifuged at 1,500 g for 8 min, and the absorbance at

freezer. The Folin–Ciocalteau method was used to measure the total

730 nm was measured using a spectrophotometer (DU 640; Beckman

concentration of phenolic compounds in the leaves (Nurmi, Ossipov,

Instruments, Fullerton, CA, USA). The standard curve was prepared

Haukioja, & Pihlaja, 1996). Two circular disks, 17–20 mm in diameter,

with six concentrations of 0-­to 50-­mg/L gallic acid, and the phenolic

were cut with a cork borer at the apical and basal parts from five fully

contents were expressed as gallic acid equivalent.

expanded and undamaged leaves. In another five leaves collected during eight samplings of Q. acutissima and eight samplings of Q. sessilifolia, the circular disk at the central part and the marginal part of both

2.3 | Sugar and water contents

the right and left sides were cut. These leaf sections were dried at

We collected leaf samples six times for deciduous Q. acutissima and

60°C overnight and then weighed to the nearest 0.1 mg. Each dried

eight times for evergreen Q. sessilifolia from May 2015 to March 2016

leaf section was powdered in a 2.0-­ml tube using a pestle and mixed

to measure the sugar and water contents of leaves. All sampling was

with 1 ml 70% aqueous acetone. The homogenate was sonicated for

performed at the study site from 10:30 to 11:00 on clear days. After

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F I G U R E   3   Seasonal changes in (a) the mean total phenolic content (gallic acid equivalent, ± SD), (b) the mean glucose content (± SD), and (c) the mean water content (± SD) in the apical and basal parts of leaves of deciduous Quercus acutissima (closed circles) and evergreen Q. sessilifolia (open circles). The number of leaves examined during each collection was n = 5, excluding those collected for glucose and water contents in May (n = 4). *p