Plant Signaling & Behavior

ISSN: (Print) 1559-2324 (Online) Journal homepage: www.tandfonline.com/journals/kpsb20

One acorn produces two seedlings in Chinese cork

oak Quercus variabilis

Jishan Xiang, Xiaolan Li & Xianfeng Yi

To cite this article: Jishan Xiang, Xiaolan Li & Xianfeng Yi (2019) One acorn produces two
seedlings in Chinese cork oak Quercus variabilis, Plant Signaling & Behavior, 14:10, e1654817,
DOI: 10.1080/15592324.2019.1654817

To link to this article: https://doi.org/10.1080/15592324.2019.1654817

Published online: 22 Aug 2019.

Submit your article to this journal

Article views: 794

View related articles

View Crossmark data

Citing articles: 2 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=kpsb20
PLANT SIGNALING & BEHAVIOR
2019, VOL. 14, NO. 10, e1654817 (4 pages)
https://doi.org/10.1080/15592324.2019.1654817

SHORT COMMUNICATION

One acorn produces two seedlings in Chinese cork oak Quercus variabilis
Jishan Xianga, Xiaolan Lia, and Xianfeng Yib
a
College of Life Sciences, Chifeng University, Chifeng, China; bCollege of Life Sciences, Qufu Normal University, Qufu, China

ABSTRACT ARTICLE HISTORY

Although the early germination of white oaks has long been considered an adaptation to counter Received 8 July 2019
animal predation, the role of the robust radicles of white oak acorns has been largely neglected in the Accepted 7 August 2019
interaction between acorns and seed-eating animals. Here, the regeneration capabilities of the pruned KEYWORDS
radicles of intermediate oak Quercus variabilis left by the acorn-eating animals were compared to test White oak; acorn; early
the ‘one acorn produce two seedlings’ hypothesis stating that the remnant radicles exhibit the capability germination; radicle;
to produce normal seedlings owning to their atypical nutritional and anatomical characteristics. Our regeneration
results first showed that both pruned radicle and cotyledons from single acorn successfully established
seedlings, providing strong evidence on an old puzzle that the robust radicles of early-germinating oak
acorns act as reserved propagules, allowing them to escape animals’ predation. Our study also suggests
that much of the investment in the food reserves of an acorn may be important as a food reward for the
animals that disperse the acorns, rather than simply serving as energy for the young seedling.

Text in the dormant plumule and cotyledonary petioles remaining

inside the cotyledons of the germinating acorns (Figure 1A).
Seedlings at the early stage of development have been recog-
This germination phenotype of acorns is very different from
nized to be the most vulnerable for the recruitment and
the white oak species from North America (e.g., Q. prinus and
colonization of various plant species.1–3 Various biotic and
Q. alba), in which the cotyledonary petioles elongate and push
abiotic factors appear to influence survival and successful
the plumule out of the cotyledons to escape predation.16
establishment of seedlings.4 Predation on the early stage of
Despite this difference, small rodents and birds actively parti-
seedlings by herbivores and granivores has been widely
cipate in pruning the cotyledons attached on the germinating
reported in various plant species,5,6 which render negative
acorns for later consuming or scatter-hoarding,8 leaving the
impacts on survival and recruitment of seedlings.7–10
robust radicles in the soil. Although the early germination of
Acorns of most white oaks germinate immediately at seed
white oak species is widely considered a general adaptation to
fall even while still attached to the trees.11,12 This rapid ger-
escape seed consumption by predators,13,14 how these germi-
mination of white oak acorns has long been termed as an
nating acorns escape animals’ predation remains unclear.
evolutionary adaptation to escape animals’ predation.13–15
Here, we investigated the regeneration capability of the
Fox13 proposed that autumn germination of white oak acorns
pruned radicles of different lengths of germinating
may result in the rapid transfer of nutritional reserves from
Q. variabilis acorns, to test the ‘one acorn produce two seed-
the cotyledons into the radicle, allowing white oak species to
lings hypothesis.’
escape predation by reducing the acorn’s attractiveness to seed
To test our hypothesis, 300 Q. variabilis acorns were
predators. In fact, the cotyledons of the germinated acorns of
planted in pots for germination experiments in the natural
most white oak species still contain substantial nutritional
conditions. Germinating acorns were categorized into three
reserves attracting seed predators,16 consequently increasing
groups based on the length of the radicles: 8–10 cm,
the probability of seedling predation by animals.2,8,17–19 These
12–14 cm, and >14 cm. We initially imitated animal pruning
biotic factors cause substantial damage to acorns or seedlings
to detach the radicles by cutting at the apical end of the acorn
of white oaks.18,20 Despite suffering from high levels of acorn
where the radicles emerge. Fifty pruned radicles of each length
and seedling predation, white oaks dominate many of the
category were planted into five pots (Diameter × Height =
temperate forests throughout the Northern Hemisphere and
40 cm × 20 cm) filled with peat soil, with ten radicles in each
show profound influences on the distribution and biodiversity
pot. Pots were cultured at room temperature. Seedling estab-
of communities as well as entire ecosystems. These facts imply
lishment was determined 8 weeks after planting. We collected
that some alternative mechanisms may function to counter
another ten germinating acorns of each length category (0 cm,
seed predation by animals in white oaks.
8–10 cm, 12–14 cm, and >14 cm), to determine the dry mass
Acorn germination of Chinese cork oak Quercus variabilis
changes of the cotyledons with regard to germination stages
and other white oaks in Asia is characterized by the elonga-
(i.e., length of radicles).
tion of the hypocotyls (0.5–1 cm) in the autumn, which results

CONTACT Xianfeng Yi yxfeng1975@126.com College of Life Sciences, Qufu Normal University, China
© 2019 Taylor & Francis Group, LLC
e1654817-2 J. XIANG ET AL.

80
Proportion of seedlings established (%)
b b
b
60

40

a
20

0
8-10 cm 12-14 cm > 14 cm
Length of the pruned radicles

c Activation Primordium of
Phloem adventitious bud
of cambium

Xylem

Phase I Phase II Phase III

Figure 1. Seedling establishment from the pruned radicles of the early germinating Quercus variabilis acorns. a: Early germination morphologies and adventitious
bud regeneration from the pruned radicles. The purple arrow points to the position where animals usually detach the cotyledons from the germinating acorns of
Quercus variabilis. The red arrows point to two seedlings regenerated from the pruned radicle and the corresponding cotyledons. b: Seedling established from the
pruned radicles of different lengths; c: Histologic sections showing the formation of adventitious buds from the pruned radicles. Data are expressed as mean ± SE.
Different letters indicate significance at P < .05 level. The letters above bars correspond to the results from multiple comparison tests.

We also collected some ungerminated acorns, germinat- 1985). The anti-nutritional tannin concentration was deter-
ing acorns with 15–20 cm long radicles, and their pruned mined using the spectrophotometry method according to the
radicles with length more than 15 cm, to measure their national standard for sorghum (GB/T15686-2008).
nutritional properties (including crude protein, crude fat, For comparison with the results obtained from the lab, 100
crude starch) and anti-nutritional tannins in the Cereal germinating acorns were labeled in an oak forest in early
Quality Supervision and Testing Center, Ministry of October 2018. Each germinating acorn was labeled by insert-
Agriculture, China. These measurements help us to know ing a 20 cm long chopstick into the soil 20 cm far away. The
how the nutrition containing in the cotyledons was trans- labeled acorns were at least 10 m apart from each along ten
ferred into the robust radicles and then promote regenera- 100 m transects to increase the probability of cotyledon prun-
tion of the radicles into normal seedlings of white oak ing by different animal species. The robust radicle of each
Q. variabilis. The protein concentration of cotyledons and germinating acorn was also carefully attached a plastic tag (2.5
taproots was measured using the semi-micro Kjeldahl appa- × 3.5 cm) using a 10-cm-long fine steel wire, to facilitate the
ratus according to the national standard for cereals and relocation of radicles if the cotyledons were pruned and
legumes (NY/T3-1982). The fat concentration was measured carried away by animals. In the next spring, we checked
according to the national standard for cereals and oil crops cotyledon predation and seedling regeneration from the
using the Soxhlet extractor (NY/T4-1982). The starch con- pruned radicles in situ.
centration was measured using the optical rotation method To see how the adventitious buds emerged from the
according to the national standard for cereals (NY/T11- pruned radicles, histologic sections were made using the

upper parts of the pruned radicles (2–4 cm downward from
the apical end of the cotyledons). The phloem, xylem, cam-
bium, and the primordium of adventitious buds were identi-
fied by optical microscopy on histologically produced sections
after safranin O and fast green staining,21 to see which part of
the radicles was responsible for the formation of the adventi-
tious buds based on anatomical observations.
Our field survey clearly showed that animals actively par-
ticipated in pruning the germinating acorns of Q. variabilis.
Of the tagged acorns, 84 were cotyledon-pruned by animals.
No difference was found in the dry masses of the pruned
cotyledons at different germination stages (F3,36 = 0.976, P =
.415) (Figure 2). However, significant differences were
observed in the concentrations of starch (F1,4 = 468.211, P <
.001), protein (F1,4 = 225.004, P < .001), and fat (F1,4 =
363.075, P < .001) between the ungerminated acorns and
those producing long radicles (Figure 3). In addition, the
radicles pruned from the germinating acorns were richer in
protein (F1,4 = 1.228E3, P < .001) but less rich in starch (F1,4 =
1.245E4, P < .001) and fat (F1,4 = 351.919, P < .001) than their
corresponding cotyledons (Figure 3). Moreover, the tannin
3 animals. No difference in the dry masses of the pruned coty-
a a a
Dry mass of cotyledons (g)
2
1 of white oak acorns.13 The rapid accumulation of anti-
0 development of oak seedlings.8,16,19
0cm 8-10 cm 12-14 cm
Length of radicles
Figure 2. Variations in dry masses and nutritional properties of cotyledons and
radicles. a: Variations in the dry masses of the cotyledons of the germinating
acorns; b: nutritional properties of cotyledons and radicles. Data are expressed as
mean ± SD. Different letters indicate significance at P < .05 level. The letters
above bars correspond to the results from multiple comparison tests.
80
Ungerminated acorns
a
Germinating cotyledons b
60
Radicles
Concentration (%)
40
c
20 c fully escape their following predation, it can be expected that
a b a b
c this process appears to be animal predation-dependent.
0
Protein Fat Starch
Chemical components
Figure 3. Variations in the concentrations of protein, fat, starch, and tannins in
the cotyledons and radicles after acorn germination. Data are expressed as mean
± SD. Different letters indicate significance at P < .05 level. The letters above
bars correspond to the results from multiple comparison tests.
PLANT SIGNALING & BEHAVIOR e1654817-3
concentration in the cotyledons and radicles of germinating
acorns was much higher than that in the cotyledons of unger-
minated acorns (F2,6 = 5.064E3, P < .001) (Figure 3).
The pruned radicles showed the capability to produce
normal seedlings in the lab, with the longer radicles exhibiting
higher capacity (F2,12 = 98.647, P < .001) (Figure 1B). In the
field, we also located 27% of seedlings regenerated from the
pruned radicles. The histologic examination showed the for-
mation of the primordium of adventitious buds due to the
activation of the intra-fascicular cambium in any of the six
vascular bundles in the radicles (Figure 1C).
Between the time of scatter-hoarding and hoarding ani-
mals’ subsequent return to recover and consume the cached
acorns, the acorns of white oak will germinate and put down
its inedible taproots into the soil.16 Therefore, food hoarding
animals must then prune the rather succulent cotyledons from
the firmly implanted taproot. In this study, we present further
evidence that animals actively participate in pruning the ger-
minating acorns of Q. variabilis. These results seem to chal-
lenge the previous prediction that the early germinating
acorns of white oak species escape predation by reducing
the acorn’s attractiveness to predators,13,19 suggesting that
other mechanisms may help white oaks escape predation by
ledons at different germination stages can still lead to a high
level of animals’ predation on the cotyledons of germinating
a
acorns. We found the radicles are much richer in nutrition
than the corresponding cotyledons, verifying the previous
prediction that a large proportion of nutrition has been trans-
ferred from the cotyledons into the radicles after germination
nutritional tannins in the cotyledons and radicles after germi-
nation is expected to counter seedling predation by animals
because seedling predation is very common for early-stage
> 14 cm We observe successful regeneration of pruned radicles into
normal seedlings both in the lab and the field. Therefore, after
the cotyledons of the acorn were excised by the food-hoarding
animals, the taproots left behind can grow into seedlings even
without the benefit of the food reserves in the cotyledons that
has consumed. These observations verified our predictions
that the robust taproots of early germinating acorns of oaks
serve as reserved propagules to produce normal seedlings in
response to animals’ predation. Importantly, different from
white oak species in North America in which both the tap-
roots and the plumules get pushed out of the cotyledons in
the fall,16 acorns of Q intermediate oak species. variabilis only
pushed the taproots rather than the plumules out of the
cotyledons (Figure 1A). Given the pruned cotyledons are
also scatter-hoarded by food-hoarding animals and success-
b c one acorn can produce two seedlings in Q. variabilis although
a
Tannins Although the germinating acorns showed no significant
differences in the dry masses of cotyledons at different germi-
nation stages (Figure 2), the nutritional contents in the coty-
ledons were decreased significantly after acorn germination.
Moreover, the radicles were found rich in protein, starch, and
fat, which provide the nutritional basis for the regeneration of
e1654817-4 J. XIANG ET AL.
the pruned radicles. However, protein may be more important
than fat and starch in supporting regeneration of the pruned
radicles. The high regeneration capacity of the pruned radicles
can also be seen from the adventitious buds at the upper parts
of the radicles. The histologic examination further showed
that the formation of the primordium of adventitious buds
can be attributed to the activation of the intra-fascicular
cambium in the radicles.
Although seed–predator interactions may be more compli-
cated than we expected in determining seedling establishment,
our results, for the first time, reveal the regeneration mechan-
isms of early-germinating oak radicles from the nutritional
and anatomical aspects. More importantly, seedling regenera-
tion from the pruned radicles in our study suggests a unique
adaptation strategy of intermediate oak (Q. variabilis) acorns
to counter seedling predation by animals, which unravels
another mechanism by which the early germinating acorns
of oak species escape animals’ predation. The reserved radicles
in other plants bearing large seeds may play an important role
in supporting seedling survival and establishment. Since most
white oak species in Asia show the same acorn germination
phenotype as Q. variabilis, we can expect that the ‘one acorn
producing two seedlings’ is an adaptive strategy for white oak
acorns to escape animals’ predation. Our study also suggests
that much of the investment in the food reserves of the
cotyledons may be important as a food reward for the animals
that disperse the acorns, rather than simply serving as energy
for the development of young seedlings.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Funding
Funding for this study was provided by the Natural Science Foundation
of Jiangxi Province [20161ACB20017, 20171BBF60073], the Natural
Science Foundation of China [31760156] and the Doctoral Starting up
Foundation of Henan University of Science and University [13480065].
References
1. Schupp EW. Seed and early seedling predation in the forest
understory and in treefall gaps. Oikos. 1988;51:71–78.
doi:10.2307/3565808.
2. Côté M, Ferron J, Gagnon R. Impact of seed and seedling preda-
tion by small rodents on early regeneration establishment of black
spruce. Can J For Res. 2003;33:2362–2371. doi:10.1139/x03-167.
3. Witmer GW, Snow NP, Moulton RS, Swartz JL. An assessment of
seedling damage by wild house mice (Mus musculus) and wild
deer mice (Peromyscus spp.). Can J For Res. 2012;42:1168–1172.
doi:10.1139/x2012-065.
4. Tambat B, Rajanikanth G, Ravikanth G, Uma SR, Ganeshaiah KN,
Kushallappa CG. Seedling mortality in two vulnerable tree species
in the sacred groves of Western Ghats, South India. Curr Sci.
2005;88:350–352.
5. Lindquist ES, Carroll CR. Differential seed and seedling pre-
dation by crabs: impacts on tropical coastal forest
composition. Oecologia. 2004;141:661–671. doi:10.1007/
s00442-004-1673-5.
6. Young HS, McCauley DJ, Guevara R, Dirzo R. Consumer
preference for seeds and seedlings of rare species impacts
tree diversity at multiple scales. Oecologia.
2013;172:857–867. doi:10.1007/s00442-012-2542-2.
7. Dalling JW, Harms KE. Damage tolerance and cotyledonary
resource use in the tropical tree Gustavia superba. Oikos.
1999;85:257–264. doi:10.2307/3546491.
8. Gómez JM, García D, Zamora R. Impact of vertebrate acorn-
and seedling-predators on a Mediterranean Quercus pyrenaica
forest. Forest Ecol Manage. 2003;180:125–134. doi:10.1016/
S0378-1127(02)00608-4.
9. Hanley ME, May OC. Cotyledon damage at the seedling stage
affects growth and flowering potential in mature plants. New
Phytol. 2006;169:243–250. doi:10.1111/j.1469-8137.2005.01578.x.
10. Abe M, Honda A, Hoshizaki K, Miguchi H. Advantage of early
seedling emergence in Fagus crenata: importance of cotyledon
stage for predator escape and pathogen avoidance. Ecol Res.
2008;23:681–688. doi:10.1007/s11284-007-0428-2.
11. Hadj-Chikh LZ, Steele MA, Smallwood PD. Caching decisions by
grey squirrels: a test of the handling time and perishability
hypotheses. Anim Behav. 1996;52:941–948. doi:10.1006/
anbe.1996.0242.
12. Smallwood PD, Steele MA, Faeth SH. The ultimate basis of the
caching preferences of rodents, and the oak-dispersal Syndrome:
tannins, insects, and seed germination. Am Zool.
2001;41:840–851.
13. Fox JF. Adaptation of gray squirrel behaviour to autumn germi-
nation by white oak acorns. Evolution. 1982;36:800–809.
doi:10.1111/j.1558-5646.1982.tb05446.x.
14. Vander Wall SB. Food hoarding in animals. Chicago:
University of Chicago Press; 1990.
15. Steele MA, Manierre S, Genna T, Contreras T,
Smallwood PD, Pereira M. The innate basis of food hoarding
decisions in grey squirrels: evidence for behavioral adapta-
tions to the oaks. Anim Behav. 2006;71:155–160. doi:10.1016/
j.anbehav.2005.05.008.
16. Yi X, Curtis R, Bartlow AW, Agosta SJ, Steele MA. Ability of
chestnut oak to tolerate acorn pruning by rodents: The role
of the cotyledonary petiole. Naturwissenschaften.
2013;100:81–90. doi:10.1007/s00114-012-0996-z.
17. Wahungu GM, Catterall CP, Olsen MF. Seedling predation
and growth at a rainforest-pasture ecotone, and the value of
shoots as seedling analogues. Forest Ecol Manage.
2002;162:251–260. doi:10.1016/S0378-1127(01)00519-9.
18. Gao XM, Du XJ, Wang ZL. Comparison of seedling recruit-
ment and establishment of Quercus wutaishanica in two habi-
tats in Dongling mountainous area, Beijing. Acta Phytoecol
Sin. 2003;27:404–411.
19. Yan XF, Zhou LB, Zhang KW, Zhou YF. Cotyledon loss and
its effects on survival and growth of Quercus wutaishanica
seedlings under different densities. Chin J Plant Ecol.
2012;36:831–840. doi:10.3724/SP.J.1258.2012.00831.
20. Yan XF, Du Q, Shi C, Zhou LB, Zhang KW. Seedling regeneration
of Quercus liaotungensis in Liupan Mountains, China. Chin
J Plant Ecol. 2001;35:914–925. doi:10.3724/SP.J.1258.2011.00914.