Flying squirrels use a mortise-tenon structure to fix nuts on understory twigs

  1. Han Xu
  2. Lian Xia
  3. John R Spence
  4. Mingxian Lin
  5. Chunyang Lu
  6. Yanpeng Li
  7. Jie Chen
  8. Tushou Luo
  9. Yide Li
  10. Suqin Fang  Is a corresponding author
  1. Research Institute of Tropical Forestry, Chinese Academy of Forestry, China
  2. College of Forestry, Hainan University, China
  3. Department of Renewable Resources, University of Alberta, Canada
  4. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, China

Abstract

Squirrels of temperate zones commonly store nuts or seeds under leaf litter, in hollow logs, or even in holes in the ground; however, in the humid rainforests of Jianfengling in Hainan, South China, we show that some flying squirrels cache elliptical or oblate nuts by hanging them securely in vegetation. These small flying squirrels were identified as Hylopetes phayrei electilis (G. M. Allen, 1925) and Hylopetes alboniger (Hodgson, 1870), in video clips captured of their behavior around focal nuts. Squirrels chewed grooves encircling ellipsoid nuts or distributed on the bottoms of oblate nuts, and then used these grooves to fix nuts tightly between small twigs 0.1–0.6 cm in diameter that were connected at angles of 25–40°. The grooves carved on the nuts (concave structure) connected with Y-shaped twigs (convex structure) and thus firmly affixed the nuts to the plant in a way similar to a mortise-tenon joint used in architecture and carpentry. Cache sites were on small plants located 10–25 m away from the closest potentially nut-producing tree, a behavior that likely reduces the discovery and consumption of the nuts by other animals. The adaptive squirrel behavior that shapes and fits nuts between twigs seems to be directed at providing more secure storage that increases food supply during dry periods in a humid tropical rainforest. In addition to providing such benefits for the squirrels, we suggest that this behavior also impacts the distribution of tree species in the forest.

Editor's evaluation

This report of nut modification and storage in flying squirrels provides new insights into food caching behaviour in wild animals. Although further direct evidence is needed to corroborate some of the findings, the current study provides valuable documentation of an interesting behaviour that should motivate further observational and experimental research.

https://doi.org/10.7554/eLife.84967.sa0

eLife digest

The rainy forests of South China are home to Cyclobalanopsis trees whose smooth, elliptical nuts are favoured by many animal species. While doing fieldwork in the Jianfengling nature reserve in the southern province of Hainan, China, researchers came across an unusual sight: many of these nuts had been wedged into the Y-shaped forks between diverging twigs. A closer inspection revealed that a carefully crafted groove on the surface of the nuts helped them to stay wedged and secured between the branches. Which creature was responsible for such a feat?

To investigate, Xu et al. set up motion-triggered, infra-red cameras near some of the hoarding sites. They discovered that the culprits were Hylopetes phayrei electilis and Hylopetes alboniger, two small species of flying squirrel that tend to store Cyclobalanopsis nuts to prepare for the dry, cool season.

The footage showed that the squirrels first chewed the nuts before inserting them tightly between the branches. In fact, this process appeared to require much care – and, potentially, cognitive involvement – with the squirrels testing and adjusting their grooves many times until a perfect fit was achieved. Caching sites were usually found 10 to 25 meters away from the nearest Cyclobalanopsis tree, which probably helps to protect the hoards from other animals on the hunt for nuts.

Squirrels from temperate regions typically prepare for winter by hiding food in the ground, between logs or inside hollow trees; in humid, tropical forests, however, such caching sites may promote mould, decomposition or germination. In these conditions, securely hanging nuts between branches may prove to be a more suitable strategy. By choosing caching sites that are away from the mother tree, squirrels may also inadvertently help Cyclobalanopsis to expand their range, with forgotten nuts becoming dislodged and sprouting in new locations across the reserve. Overall, these findings shed new light on animal adaptation and cognition, as well as on the forces that help to shape forest ecology.

Introduction

Storing food to buffer against periods of low resource supply is a common species-specific behavior used by squirrels and other rodents (Andersson and Krebs, 1978; Steele et al., 2006). Nuts, in particular, are harvested from trees and cached in various places. For example, many temperate-zone squirrels hoard nuts under leaf litter, in holes in trees or logs or in the ground (Cheng et al., 2005; Hadj-chikh et al., 1996). In subtropical zones, however, some species store nuts or mushrooms by hanging them on tree branches, a behavior thought to minimize fungal infection in humid environments (Lichti et al., 2017; Xiao et al., 2013) or decrease the risk of loss through decomposition or germination under warmer temperatures in the cache (Sechley et al., 2015).

The present work was prompted by our inadvertent discovery of Cyclobalanopsis nuts with strange surface grooves, and that were suspended in Y-shaped crotches of twigs on understory plants on Hainan Island, South China. Cyclobalanopsis trees are dominant fagaceous trees in these tropical forests; however, their fruits are elliptical or oblate single nuts with smooth surfaces, features that make them difficult to hang on vegetation. Firmly suspending such nuts in the vegetation presents an ecological challenge to squirrels in such environments. We asked whether some of the nine squirrel species identified from Hainan forests used special behaviors to prepare these nuts and fix them securely on vegetation.

In this paper, we show that the Indochinese Flying Squirrel, Hylopetes phayrei electilis (G. M. Allen, 1925), and the Particolored Flying Squirrel, H. alboniger (Hodgson, 1870), which co-occur in Hainan Island, cache these nuts individually between the twigs of small plants. Both of these small-bodied flying squirrels are widespread in the tropical forests in Southeast Asia, from Myanmar, south to northwestern Vietnam, and east into southern China (Duckworth et al., 2016; Duckworth et al., 2016). In China, H. phayrei electilis can be found in the mountainous areas of Hainan, Fujian, Guangxi, and Guizhou Provinces. H. alboniger is mainly found in the provinces of Hainan, Yunnan, Guizhou, Guangxi and, more rarely, in Zhejiang.

Although these squirrels are reasonably common, there is little published information about their habits, and there are no studies from Hainan Province in China (Li et al., 2012). In particular, nut storage behavior hasn’t been reported from elsewhere in the ranges of either of these two squirrels. Thus, the preparation of nuts to connect them firmly to twigs is a new finding, although other squirrel species are known to handle nuts prior to suspending them to improve the success rate of storage (Fox, 1982; Steele and Yi, 2020; Xiao et al., 2010). Here, we document in some detail the squirrel behaviors associated with this phenomenon in Hainan.

Results and discussion

We used images from infrared cameras to determine that the nocturnal flying squirrels, H. phayrei electilis and H. alboniger, two of the nine species of squirrels known from tropical forests of Hainan (Table 1), stored Cyclobalanopsis nuts by suspending them on vegetation in the Jianfengling forest (Videos 15). The videos further showed that the squirrels chewed grooves in the surfaces of the nuts before fixing between the twigs, and that they sometimes altered the previously carved grooves by further chewing, apparently to adjust the fit and suspend the nut more firmly (Videos 69). In footage from 32 field infrared cameras, we captured 48 film sequences that included chewing (Videos 67), fixing (Video 9, partial evidence) and removing nuts (Video 2, Video 3 and Video 5), or visiting a storage site (Video 1, Video 4 and Video 8). This direct evidence, together with the findings below, shows that this mode of nut storage is a reasonably common activity of these two squirrel species in the Jianfengling forest.

Table 1
The nine squirrel species known from Jianfengling, Hainan Island, China.
Species and subspecies nameBody length/mm
Tamiops maritimus (Bonhote,1900) (Liu et al., 2020; Pan et al., 2007)105~134
Dremomys pyrrhomerus (Thomas, 1895) (Xu and Chen, 1989)194~215
Hylopetes alboniger (Hodgson, 1870) (Liu et al., 2020; Andrew, 2008; Pan et al., 2007)180~203
Hylopetes phayrei electilis (Allen, 1925) (Liu et al., 2020; Pan et al., 2007)123~173
Dremomys rufigenis (Blanford, 1878) (Zheng et al., 2008)170~250
Callosciurus erythraeus (Pallas, 1779) (Huang, 1995; Zheng et al., 2008)198~252
Belomys pearsonii (Gray, 1842) (Pan et al., 2007; Huang, 1995)180~260
Petaurista albiventer (Gray, 1834) (Jing et al., 2007)420~520
Ratufa bicolor (Sparrmann, 1778) (Li et al., 2008)350~505
  1. Note: The data in this table are referenced from the below literature.

Video 1
Squirrel 1 of Hylopetes alboniger was checking and re-fixing nuts at the storage sites with footage from infrared cameras.
Video 2
Squirrel 2 of Hylopetes alboniger was removing nuts from storage sites with footage from infrared cameras.
Video 3
Squirrel 3 of Hylopetes alboniger was removing nuts from storage sites with footage from infrared cameras.
Video 4
Squirrel 4 of Hylopetes phayrei electili was checking and re-fixing nuts at storage sites with footage from infrared cameras.
Video 5
Squirrel 5 of Hylopetes phayrei electili was removing nuts from storage sites with footage from infrared cameras.
Video 6
One squirrel was cracking the nuts on the ground with footage from infrared cameras.
Video 7
One squirrel was rotating and cracking the nuts on the trees with footage from infrared cameras.
Video 8
One squirrel was re-fixing the nuts by cracking behavior with footage from infrared cameras.
Video 9
One squirrel was fixing the nuts between the twigs with footage from infrared cameras.

We merged several photos and a video successively taken by an infrared camera in 30 s.

A total of 151 grooved and cached nuts were found suspended on more than 55 tree or shrub species distributed across 28 plant families during our censuses of approximately 5.5 ha of forest (Figure 1, Supplementary file 1). All suspended nuts found had surface grooves of the form carved by squirrels, as documented above. Examples of storage locations and carved nuts are shown in Figures 23. Most discovered nuts were fixed between plant twigs connected at angles of 25–40° on a variety of small saplings and shrubs (Figure 4). This range of angles accommodates the nut sizes of Cyclobalanopsis edithiae and C. patelliformis (2.4 cm (width) × 4.6 cm (length) and 2.4 cm (width) × 2.0 cm (height), respectively), which accounted for 96.7% of the nuts that we found cached (C. edithiae (40.4%), C. patelliformis (56.3%)). A few nuts of Lithocarpus fenzelianus A. Camus (n=4) and C. fleuryi (Hickel & A. Camus) Chun ex Q. F. Zheng (n=2) were also found similarly suspended on plants.

Spatial distribution of the 151 suspended nuts observed in Jianfengling Nature Reserve, Hainan, China.
Nuts are stored after surface preparation by flying squirrels.

(A) Nut of C. edithiae (Skan) Schottky, with chewed grooves outlined in red. Nuts of C. edithiae fixed on trees, with (B–D) one groove, (E) two non-connected grooves, or (F) spiral carved grooves encircling the nuts. (G) Nut of C. patelliformis (Chun) Y. C. Hsu et H. W. Jen, with chewed grooves outlined in red. (H–I). Nuts of C. patelliformis fixed on trees, with carved grooves on the bottom fixed on (J) bamboos, (K–L) lianas, between the big petioles of (M) trees and (N) palms.

Variation in carved grooves depends on the storage situation.

The carved surface grooves on nuts of C. edithiae mostly encircle the middle of the nut, with (A) one groove, (B) one spiral groove, or (C) two separated grooves. The grooves on nuts of C. patelliformis are distributed on the bottom of the nuts, with (D) 2, (E) 4, (F) 6, (G) 8, (H) 10 symmetrically, or (I) randomly distributed grooves.

Nuts were fixed tightly between twigs generally meeting at angles of 25–40°.

(A) C. edithiae nuts. (B) C. patelliformis nuts.

Nuts of the two predominant tree species were disproportionately stored on small plants with diameters at breast height (DBH) of 0.4–1.6 cm (Figure 5) and twig diameters of 0.10–0.60 cm (Figure 6a and b). For nuts of C. edithiae, plant twig diameter was significantly correlated with groove width on the nut, and generally varied from 0.20–0.60 cm (p<0.001, Figure 7). The widths of grooves carved in the nuts matched the typical width of the paired incisor tips of these squirrels (i.e. less than 5 mm). Most of the nuts were found stored on the first to third branches of a plant 1.50–2.50 m above the ground (45.9% of C. edithiae and 43.5% of C. patelliformis storage sites) (Figure 8).

Most nuts were stored on small plants with the diameter at breast height (DBH) ranging from 0.4–1.6 cm.

(A) C. edithiae nuts. (B) C. patelliformis nuts. Notes: The value on each bar is the actual number of stored nuts.

Nuts were stored mainly on small plants between twigs with diameters of 0.10 - 0.60 cm.

(A) Histogram of diameters of twigs used to store nuts of C. edithiae. (B) Histogram of diameters of twigs used to store nuts of C. patelliformis. Notes: The value on each bar is the actual twigs with the number of stored nuts.

Grooves carved by squirrels on most C. edithiae nuts were 0.2 - 0.6 cm in width.
Nuts were generally stored on the first to third branches at 1.5–2.5 m aboveground.

(A) C. edithiae nuts. (B) C. patelliformis nuts. Notes: The value on each bar is the actual number of stored nuts.

Squirrels of both species carved spiral zigzagged grooves that encircled the midsection surface of the ellipsoid nuts of C. edithiae (Figure 2a–f) with one, or occasionally, two grooves (Figure 3a–c). Two non-connected or spiral grooves appeared to be useful for adjusting the position of stored nuts to the specific orientation of the twigs. In contrast, up to 20 surface grooves were carved on the bottoms of the oblate nuts of C. patelliformis (Figure 2g–i). These grooves on oblate nuts varied considerably in pattern from symmetric (Figure 3d–i) to scattered. Symmetrical grooves on the bottom of nuts likely facilitate firm positioning as squirrels rotated nuts, apparently to optimize the nut’s position for the most secure attachment.

Interestingly, oblate nuts stored on living trees and shrubs had significantly more carved shallow scattered grooves than those stored on dead trees and lianas (5.1 ± 5.0 vs 2.8 ± 4.0, t=2.1591, df=46.402, p=0.036). Because the bark of dead trees and lianas is coarser than that of living trees, fewer grooves may be required to hold the nuts securely in place. We also note that the grooves on the ellipsoid nuts of C. edithiae were deeper (more than 0.5 mm) than those on the oblate nuts of C. patelliformis (less than 0.45 mm, p<0.05, Figure 9). Nonetheless, none of the chewed grooves that we observed were deep enough to damage the endosperm of the nut, and thus the squirrels seemed to minimize the potential impacts of fungi during storage.

Number of grooves carved on the oblate nuts of C. patelliformis.

(A) Most nuts had fewer than eight grooves. (B) The depth of most grooves was shallow to medium. Notes: The value on each bar is the actual number of stored nuts.

The surface grooves allowed the squirrels to ‘pressure fit’ the nuts between the two plant twigs in a way functionally similar to a mortise-tenon joint (Qiao et al., 2021; Figure 3). Squirrels used the twigs as a convex ‘tenon’ to fit into the convex ‘mortise’ provided by the grooves on the nuts. Thus, carved nuts were inlayed between plant twigs (0.10–0.60 cm in diameter) intersecting at specific angles (25–40°) on various understory plants (Figure 4). We found nuts on small trees and shrubs, but also on lianas, bamboos, or dead trees, and even occasionally on large petioles of palms or trees (Figure 2j–n, Table 2). Once fixed in this manner, nuts were resistant to being blown off by strong wind or even by shaking that we administered experimentally (Videos 1015).

Table 2
The types of plants used for nut storage.
Plant typeNumber of individualsPercentage of all individuals (%)
Alive tree10871.5
Dead tree1711.3
Alive liana1912.6
Dead liana21.3
Bamboo53.3
Total151100
Video 10
Footage of shaking a liana does not dislodge nuts of Cyclobalanopsis edithiae stored by squirrels.
Video 11
Footage of shaking a sapling does not dislodge nuts of Cyclobalanopsis edithiae stored by squirrels.
Video 12
Footage of shaking a sapling does not dislodge nuts of Cyclobalanopsis edithiae stored by squirrels.
Video 13
Footage of shaking a liana does not dislodge nuts of Cyclobalanopsis patelliformis stored by squirrels.
Video 14
Footage of shaking a sapling does not dislodge nuts of Cyclobalanopsis patelliformis stored by squirrels.
Video 15
Footage of shaking a sapling does not dislodge nuts of Cyclobalanopsis patelliformis stored by squirrels.

The distance between the closest Cyclobalanopsis trees producing nuts and storage sites on smaller understory plants varied from 10–25 m (Figure 10), distances greater than the average canopy width of large trees in the Jianfengling forest (estimated to be 10 ± 5 m). This sort of distancing likely reduces discovery by other squirrels, mice, or other animals potentially searching for aboveground nuts below the parent trees (Cao et al., 2011), although recordings from our cameras show that some nuts were still found and eaten by mice.

Distance from storage sites to potential parent trees for the nuts varied from 10–25 m.

(A) C. edithiae nuts. (B) C. patelliformis nuts. Notes: The value on each bar is the actual number of stored nuts.

Because of this spacing, most seedlings that result from dropped, fallen or forgotten nuts (Figure 11) will germinate at some distance from their parents. Thus, seed dispersal by these squirrels may decrease competition between seedlings and parent trees. This should increase seedling survival rates and could, in turn, truly decrease the negative density dependence of conspecific trees (see Detto et al., 2019). Unfortunately, we presently do not have sufficient data to estimate what proportion of the disappearance that we observed is the result of use by squirrels, although our video footage establishes that they do remove some nuts (Videos 25). Nonetheless, some proportion of nuts likely falls from storage sites and germinates nearby, as is common for seeds and nuts cached by squirrels, especially in hardwood forests (Steele and Yi, 2020).

After long (e.g. >ca. 365 days) storage, nuts become not fresh.

(A) Dried nuts, (B) Germinated, or (C–E) Destroyed by insects.

Only 63.6% of nuts that we discovered on understory plants were fresh at the time of the survey. Under natural conditions, these nuts on the ground would likely germinate in ca. two to three months after they had fallen to the ground (Zhou, 2001). These stored nuts did not germinate during our 3.5 month investigation interval, which means that nuts can persist in these storage sites for longer periods than do nuts on the ground. Over the 44 days between the first and second surveys, 19.7% of the stored nuts had disappeared, and 15.0% of the nuts discovered during the second survey were new. Over the 61 days between the second and third surveys, 43.7% of the stored nuts had disappeared, and 20.6% of the nuts discovered were new. Thus, the numbers and composition of stored nuts in this forest are dynamic variables.

In general, the number of nuts being stored decreased gradually from January to May after the fruiting season, i.e., it decreased gradually through the dry season toward the rainy season. The high precipitation and humidity of the Jianfengling forest environment (Xu et al., 2015) likely favors the storage of nuts above the ground by reducing fungal infection or germination and may influence the timing of removal of stored nuts. In more temperate forests with lower annual precipitation nuts can be safely stored under dry leaf litter or in the ground without special processing (Hadj-chikh et al., 1996). Hence, the suspended storage that we observed appears to be an effective adaptation for safe storage, mainly during drier seasons to improve the food supply for the squirrels during the colder months at Jianfengling. However, field comparative data about the fates of seeds stored on or above are needed to properly evaluate this hypothesis.

In summary, we have demonstrated that individuals of the flying squirrel species H. phayrei electilis and H. alboniger collect and cache nuts from or beneath two species of Cyclobalanopsis trees in the Jianfengling forest. Nuts were carried 10–25 m away from parental fruiting trees and processed for storage by chewing grooves into their surfaces before they were suspended on shrubs or small trees (Videos 67). The pattern and depth of these grooves varied with the shape of nuts from these two Cyclobalanopsis trees so that they could be effectively fixed in the crotches of two twigs. Squirrels appear to check the strength of fixation, and sometimes iteratively modify the grooves to improve the attachment (Video 1, Video 4 and Video 8) before a nut is finally removed from a storage site (Video 2, Video 3 and Video 5).

Clearly, individuals of these two squirrels store nuts of different shapes and sizes securely on a variety of plant twigs at some distance from the plants that produced the nuts. The behavior of these two squirrel species has evolved to prepare nuts for such storage by chewing surficial grooves on nuts to enable a ‘mortise-tenon’ connection between nuts and understory plant twigs. The significant efforts that we observed of squirrels testing and adjusting the fixation of nuts suggest that they employ active cognitive processes in storing these nuts.

Taken together, our observations suggest that effective food storage behavior is a significant aspect of the adaptation of these two flying squirrel species to life in the humid tropical rainforest. Nut caching behavior helps to secure food for the coolest month in these rainforests. We have not yet compared the fates of suspended nuts with those of the same species buried in the ground or leaf litter, but predict that suspended storage will be far superior. The caching behavior may further affect the dispersal of nuts (Chang and Zhang, 2014; Xiao et al., 2004) in a way that alters the spatial and temporal distribution of the local plant community in the long run. Thus, this behavior could have a significant impact on the larger forest community. Although the importance of large DBH trees has been emphasized for the maintenance of forest ecosystem productivity (Lutz et al., 2018); however, from a broader perspective that includes the understanding of squirrel caching behavior, small understory plants may help sustain the diversity and complexity of forest structure. The possibility that such plant-animal interactions affect tree populations and distributions deserves more attention in the future (Goheen and Swihart, 2003; Rong et al., 2013).

Materials and methods

Study site

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This study was conducted in the Jianfengling region of the Hainan Tropical Rainforest National Park in Hainan Province, China (108°46'–109°45'E). The area has a seasonal tropical monsoon climate with a rainy season from June to October and a dry season from November through May of the next year. The mean annual temperature in this forest is 19.7 °C and the annual average precipitation is 2461 ± 619 mm. Jianfengling is the second rainiest area on Hainan Island, with an average annual relative humidity of >88% (Jiang and Lu, 1991).

The Jianfengling forest includes 992 free-standing tree and shrub species, and is dominated by trees of Fagaceae, Lauraceae, and Moraceae (Xu et al., 2012). Castanopsis, Lithocarpus, and Cyclobalanopsis are the three main genera of Fagaceae, which reproduce through nuts that are used as food by various mammals. The cupules of Castanopsis are solitary units produced on a rachis, completely or partially enclosing the nut, while cupules of Lithocarpus are grouped together in cymes on the rachis, completely or partly enclosing the nut. In contrast, cupules of Cyclobalanopsis are solitary, and do not enclose the nuts. Because enclosed nuts are difficult for squirrels to deal with, Cyclobalanopsis nuts are highly preferred as food by squirrels and other animals, although the smooth nut surfaces make them challenging to suspend on vegetation. Cyclobalanopsis edithiae (Skan) Schottky and Cyclobalanopsis patelliformis (Chun) Y. C. Hsu et H. W. Jen are the two most abundant species with naked nuts in the mountain forests of Jianfengling (Xu et al., 2015; Table 3). Both are in fruit from October to December, just before the coolest month (January) in Hainan.

Table 3
Main Fagaceae species found in a 60 ha plot in the Jianfengling forest.
SpeciesAbundance
Castanopsis carlesii (Hemsley) Hayata3269
Castanopsis fissa (Champion ex Bentham) Rehder & E. H. Wilson2803
Castanopsis jianfenglingensis Duanmu2297
Castanopsis tonkinensis Seemen953
Castanopsis ledongensis C. C. Huang & Y. T. Chang335
Castanopsis fabri Hance113
Castanopsis hystrix J. D. Hooker & Thomson ex A. de Candolle35
Cyclobalanopsis edithiae (Skan) Schottky1645
Cyclobalanopsis patelliformis (Chun) Y. C. Hsu & H. W. Jen1207
Cyclobalanopsis phanera (Chun) Y. C. Hsu & H. W. Jen886
Cyclobalanopsis fleuryi (Hickel & A. Camus) Chun ex Q. F. Zheng568
Cyclobalanopsis neglecta Schottky392
Cyclobalanopsis blakei (Skan) Schottky279
Cyclobalanopsis hui (Chun) Chun ex Y. C. Hsu & H. W. Jen220
Lithocarpus longipedicellatus (Hickel & A. Camus) A. Camus2842
Lithocarpus pseudovestitus A. Camus2427
Lithocarpus fenzelianus A. Camus1751
Lithocarpus amygdalifolius (Skan) Hayata1360
Lithocarpus handelianus A. Camus1046
Lithocarpus fenestratus (Roxburgh) Rehder323
Lithocarpus howii Chun130
Lithocarpus hancei (Benth.) Rehd.71

Field investigation

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During work in the Jianfengling forest, we discovered Cyclobalanopsis nuts with surface grooves tucked into the Y-shaped crotches of twigs on understory plants (Figure 2). The regular grooves showed signs of having been chewed by some unknown animal(s) (Figure 3), perhaps in order to increase the friction between nuts and plant twigs so as to fix the nuts securely in place. Thus, we conducted a systematic field investigation from January to May 2022 to discover the animals involved and to study their nut caching behavior in more detail.

We first made a systematic search of ca. 5.5 ha of forest for grooved nuts suspended in vegetation to seek relationships between this phenomenon and various plant types. We used plastic tags to mark all plants discovered bearing grooved nuts. The species identity, diameter at breast height (DBH), geographical position, and elevation of plants found with suspended grooved nuts were recorded. We also measured the diameter and angles of the two twigs where the nuts were fixed. Identities of trees with DBH ≥20 cm within a ca. 20 m radius of each stored nut were recorded. Finally, we measured the distance between the storage site and the nearest tree where the nuts could have been produced. Additionally, we recorded the species, weight, diameter, and height of each plant for all suspended nuts discovered. Number, depth, and position of the surface grooves were also noted for each nut. Because precise measurement was impossible, the depth of the carved grooves was measured as a categorical variable classified in the following three groups: shallow (ca. 0–0.15 mm), medium (ca. 0.15–0.30 mm), and deep (ca. 0.30–0.45 mm). We also recorded whether each nut was fresh, eaten by insects, or infected by fungi; fresh nuts are green but become wrinkled and black with age. All nuts were photographed at the time of discovery.

The first search for cached nuts was carried out on January 15, 2022. We re-surveyed the site 44 days later on February 28, using the same protocols described above, to learn whether the nuts recorded previously were still where we initially found them, and to find any new nuts that had been stored in the area. A third similar survey was made 61 days later on April 30.

Because we initially knew neither the identity of the animals that stored the nuts nor how nuts were stored and retrieved, we set up 32 motion-activated infrared cameras (22 WildINSights 20MP 1080 P HD Trail Cameras and 10 WildINSights 5MP 960 P HD Trail Color Cameras) around the stored nuts to monitor animal activities that might be related to nut storage or consumption. These cameras were positioned to view both typical nuts that we found and their surroundings. In general, the distance between a camera and a focal nut was 0.5–1 m. Animals filmed as being associated with the nuts were subsequently identified to species by experts using the resulting pictures and videos. Especially, for Video 9, we merged several photos and a video successively taken by an infrared camera in 30 s near the 64 ha permanent plot in Jianfengling, Hainan on January 23, 2023.

Statistical analyses

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We described the distribution twig angles which were used to fix the nuts and twig diameters as histograms. Standard t-tests were used to assess the significance of differences between paired sets of variables, and linear regression was used to assess relationships between the sizes of the two twigs that constituted each nut storage site. Histograms were also drawn to describe variations in the sizes and location of storage plants and carved nuts, including the width of grooves carved by squirrels, DBH and height of plants with stored nuts, number of carved grooves of each nut, and distance of stored nuts to the nearest fruiting tree of that species.

We also used t-tests to study whether the oblate nuts stored on living trees and shrubs had significantly more carved shallow scattered grooves than those stored on dead trees and lianas, and whether grooves on the ellipsoid nuts of C. edithiae were deeper than those on the oblate nuts of C. patelliformis.

Data availability

All data are available in the main text or the supplementary files.

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    8. Yang H
    9. Zhou Z
    (2015)
    Jianfengling Tropical Mountain Rain Forest Dynamic Plot: Community Characteristics, Tree Species and Their Distribution Patterns
    Beijing: Chinese Forestry Publishing House.
  8. Book
    1. Zheng ZM
    2. Jiang ZK
    3. Chen AG
    (2008)
    Rodents Zoology
    Shanghai: Shanghai Jiao Tong University Press.
  9. Book
    1. Zhou T
    (2001)
    Cultivation Techniques of Main Tropical Economic Trees in China
    Beijing: Chinese Forestry Publishing House.

Decision letter

  1. Ammie K Kalan
    Reviewing Editor; University of Victoria, Canada
  2. Christian Rutz
    Senior Editor; University of St Andrews, United Kingdom
  3. Ammie K Kalan
    Reviewer; University of Victoria, Canada
  4. Pizza Chow
    Reviewer; University of Chester, United Kingdom

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Smart squirrels use a mortise-tenon structure to fix nuts on understory twigs" for consideration by eLife.

Your article has been reviewed by three peer reviewers, including Ammie K Kalan as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Christian Rutz as Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Pizza Chow (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this decision letter to help you prepare a revised submission.

Essential revisions:

All three reviewers found this work interesting and novel, but agreed that the claims need to be significantly tempered given the data at hand. When revising the article, please pay attention to the detailed comments raised in the three reports, which are appended below, but ensure you address the following three major concerns:

1) Since no squirrels were actually observed creating the grooves, the whole article needs to be refocused on the recovered artefacts and observed behaviours (caching etc.). This will require a new title and substantial text revision. It should be made clear throughout that the authors have no data at present to show that flying squirrels actually made those grooves (although we would welcome any new data to support this claim, such as incisor matching mentioned by Reviewer #2). Ideally, this would be added to a section titled "Limitations of the study".

2) Related to the point above, all speculation about the evolution of this behaviour in flying squirrels and the potential underlying cognitive mechanisms should be removed from the article, since the data are simply too preliminary at present to address these points.

3) If possible, the authors should consider a more robust analysis of nut retention in the trees, with and without grooves, or a comparison of how nuts fare on the ground versus in the trees, to support their arguments (see also comments by Reviewer #3).

There is also scope for polishing the presentation, and we recommend a thorough language check.

Finally, please note that eLife has adopted the STRANGE framework, to help improve reporting standards and reproducibility in animal behaviour research. In your revision, please consider scope for sampling biases and potential limitations to the generalisability of your findings:

https://reviewer.elifesciences.org/author-guide/journal-policies

https://doi.org/10.1038/d41586-020-01751-5

Reviewer #1 (Recommendations for the authors):

I really enjoyed reading this paper and I commend the authors on the great detail with which they have recorded the behaviour. I have a few suggestions to make that I think would help to improve clarity and support to some of the text as it currently stands.

line 72-73: this sentence does not make sense, please rephrase.

line 96-97: expand upon what "actively associated" means here, i.e., just briefly state some of the behaviours observed since this is key.

You never see the squirrels actually making the grooves but it is assumed, so I think you need to clearly state this to the reader.

Can you tell us how many unique individuals were seen in your videos? at least a minimum number?

line 129: after forest, do you have a reference you can cite?

line 163: we hypothesize, rather than guess… this is also an example of a sentence needing rephrasing due to grammar issues (see last point).

line 165: I think you could clarify what learning ability you are talking about. At the moment this sentence is confusing because it seems like you are stating this is potentially tool use? It would be good to be more clear what you mean here and perhaps to also clearly state that this isn't tool use, but again to focus on or better explain what aspects of clever cognition this behaviour is evidence of.

line 168: why animal cranial zootomy? In general I could not follow your thinking in this sentence.

line 196: reference for sentence ending with 88%?

line 224: what do you mean by dominant trees?

Figure 2 caption: A) one groove (?) – the word groove is missing I think.

Figure 3: although I find the angles very interesting I think the text fails to make clear why the angle might matter. Could you include in the text for example a clear explanation of why twig angles 25-40 degrees are most frequent for this behaviour?

Figure 4 caption: I think maybe missing some words between lines 403 and 404 or else not clear

Reviewer #2 (Recommendations for the authors):

A bit more development of the evidence for cache loss through decomposition ('hoard rot' as it is sometimes referred) or germination and the environmental correlates of that would be helpful earlier on for the reader to understand the observations you present.

Overall the paper would benefit from some copy editing for clarity, as some sentences are a bit difficult to follow in their current form.

I have two concerns about attributing the caching to the squirrels that could be resolved by the following:

1. Direct observation of the squirrels performing this behavior.

2. Incisor matching – do the grooves carved out of the nuts match the width of the typical width of the two incisors of these species?

3. Consideration of the following: why would an arboreal species, such as a flying squirrel, cache nuts only on the lower branches of these plants, rather than higher up? That these behaviors only appear to be at eye-level to humans does make me question why squirrels would limit themselves to this height as well.

Line specific suggestions:

Line 25 – the use of 'evolutionary challenge' – I would argue it is more an ecological challenge.

Line 29 – specify what nation/region Hainan Island is in.

Line 33 – the mortise and tenon joint has a long history outside China as well; it may be more recognizable to readers to briefly articulate what the joint is used for/what it looks like rather than specify a nationality.

Line 52 – not clear what species of nuts are being discussed here, so the comment that they are in clusters doesn't immediately register. Clustering is perhaps a more species-specific phenomenon (most of the nuts I am familiar with in a temperate zone do not cluster), so this requires more explanation here.

Line 56 – again, not sure this is necessarily an evolutionary challenge but an ecological one.

Line 58 – Write out examples here. The Hunt et al. paper doesn't seem applicable here as it focuses on learning in locomotion, rather than cognitive food handling decisions. I would also avoid the use of the term 'smart' in discussing animal cognition; it is to me more of an anthropomorphic term and isn't necessary to describe the behaviour observed here, but I acknowledge this may be more of a personal preference.

Line 54 – 'most' nuts – would like to see some data to back this up, or at least examples of species that are commonly harvested by squirrels and therefore relevant here.

Line 59 – is there evidence that squirrels use tools elsewhere?

Line 68 – I would modify this – the behavior hasn't been reported elsewhere (have you checked databases of nature observations to be sure?)

Line 69 – Perhaps not to facilitate the physical lodging of a nut into a crevice, but other species of squirrels do indeed handle nuts prior to caching which improves the success rate of storage (see for example Fox 1982, Steele et al. 2001, Xiao et al. 2010).

Supplementary Figure 1 would be helpful to have in the main text.

Figure 1 – I think providing a visual comparison to the type of joint you are likening these carvings to would be helpful here, because these nut carvings appear quite different than what I would have expected given what my understanding of the mortise-tenon joint is.

Line 132 – what is the criterion for "fresh"? – OK, I see now in the methods further down.

Line 133 – how were nuts marked in the first survey to enable these conclusions?

Paragraph at Line 163 – this is very speculative; which given that this is more of a natural history observation, is fine, but it could be much more developed in order to put this study into context and to demonstrate its value. Evolution of learned behaviors that enhance survival and reproduction is a rich topic in animal behavior, and evolution of food caching behaviors in particular has received significant attention. Feeding innovations and their relation to behavioral evolution are important (as you note, tool use in corvids and parrots are excellent examples), and perhaps describing these observations against that background with citations to the literature would help make sense of this story.

Line 170 – This is late in the paper to introduce the fact that there are nine species in this region – this should be moved up.

Line 173 – have they evolved this behavior? Or is it learned or innovated? Evolution is a bold claim in the absence of familial data including fitness metrics and given how tightly clustered in time and space these observations are.

Line 180 – this is also a 'just-so' fallacy; you observed the persistence of these nuts over a very short time and did not present controls of what happens to these same species of nuts should they be stored on the ground or in the leaf litter. My experience in the tropical rain forest leads me to believe that this behavior is highly likely to have that effect of cache protection, but just because it makes logical sense does not make it data.

Reviewer #3 (Recommendations for the authors):

The authors described and documented a nut-storing behaviour of two species of flying squirrels that is not previously mentioned in the literature. The authors found that the two species of flying squirrels made grooves in nuts so that they can be fixed between twigs of different species of plants and shrubs. The authors conclude that the squirrels of these populations are evolved or learn to do so, as well as placed such behaviour in tool-use context and claim that they are 'smart'.

Overall, I enjoyed reading the manuscript, and knowing more about squirrels' behaviour. I also find the work is important, because there isn't proper documents stating this nut grooving behaviour in squirrels. This will spark an interest of understanding the causes and consequences of this nut grooving behaviour, which advances different fields such as animal behaviour and cognition, behaviour ecology, evolution and ecology.

Despite the strengths of the study, I do have several suggestions to improve the manuscript:

1) One of the goals of the study is to show that these squirrels are smart. Now I am not saying that they are not, but we need proper evidence, systematic investigations, and analyses to establish this claim. For now, these claims are not entirely supported by analyses or proper measurements; the definition of 'smart' and relating nut grooving behaviours to tool use is not complete. For example, no evidence indicating this behaviour involves complex cognition or cognitive process – smart, or it is related to fast learning performance – smart. All we know is the squirrels did it, but how they acquire such skills and whether it involves in complex or advance cognition is another story. With this in mind, many wordings appear to lead readers think that this is established, which is not the case.

2) Analyses – the current analyses basically is 0. Can't the authors pay a little more efforts in analyses, say using (M)ANOVA and post hoc tests to examine whether the number of as well as the depth of grooves differ in nuts that store on ground and (live and dead) trees? Or depending on the shape of a nut too? More importantly, out of how much footage did the authors capture the flying squirrels did the nut grooving behaviours? what other animal species interacted with those nuts? These are the central aims of the manuscript but are not done properly.

3) The current presentation of the manuscript is largely oriented to plants, and less about animal behaviour (definitely not involved in animal cognition). This appears to be a bit contradictory to what the authors would like to present or what they found interesting; that the grooving behaviour of seeds in flying squirrels is novel. Suggest thinking about how to reorganise this information and focus on animal behaviour

4) Figures – please present proportion or percentage on y axis, place the actual value on top of each bar in the bar graph; this will make information more presentable as well as increase the transparency of data.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your article entitled "Flying squirrels use a mortise-tenon structure to fix nuts on understory twigs" for further consideration by eLife. Your revised article has been evaluated by Christian Rutz (Senior Editor) and a Reviewing Editor (Ammie Kalan).

The article has been much improved but there are some remaining issues that need to be addressed, as outlined below:

1) We appreciated the addition of the new videos to demonstrate the squirrels' active interaction with the nuts, but could you please address Reviewer #3's alternative explanation directly in the text and provide any further clarification regarding the carving or cracking behaviour?

2) Similarly, please modify the text to be explicit about which parts of the behaviour you have direct video evidence for and which actions are inferred and, therefore, still require further supporting evidence. This will improve transparency and help readers evaluate your findings.

3) Please address the remaining comments from the reviewers below.

Reviewer #3 (Recommendations for the authors):

Thank you very much for inviting me to review this paper again. I also thank the authors for making a great effort to revise the paper, and most of my previous suggestions/concerns have been taken into the revision. The revised paper is still interesting as it documents the grooved nut in between twigs that may be made by this species of squirrel (a behaviour that is not fully understood).

In the past version of the manuscript, one of the main weaknesses of the paper was the absence of evidence showing squirrels made grooves and fixing them in between the twigs. In this version, the authors submitted three new videos (videos 6-8), and they are useful for seeing this species of squirrel handling a nut. However, videos 6 and 7 are showing a squirrel rolling a nut and attempting to 'carve' them and Video 8 is a squirrel finding a nut and attempting to take it out between the twigs. The terminology 'carving' is debatable, as to some squirrel experts, this appears to be 'cracking' the nut shell instead of 'carving'. None of these videos really shows direct evidence as in a squirrel grooving a nut and fixing it in between the twigs. If the authors insist that the squirrels do so (which from line 92 appears to be the case), we would need videos that show a squirrel making these grooves and fitting the nut in between twigs.

https://doi.org/10.7554/eLife.84967.sa1

Author response

Essential revisions:

All three reviewers found this work interesting and novel, but agreed that the claims need to be significantly tempered given the data at hand. When revising the article, please pay attention to the detailed comments raised in the three reports, which are appended below, but ensure you address the following three major concerns:

1) Since no squirrels were actually observed creating the grooves, the whole article needs to be refocused on the recovered artefacts and observed behaviours (caching etc.). This will require a new title and substantial text revision. It should be made clear throughout that the authors have no data at present to show that flying squirrels actually made those grooves (although we would welcome any new data to support this claim, such as incisor matching mentioned by Reviewer #2). Ideally, this would be added to a section titled "Limitations of the study".

We have included three additional videos to demonstrate that squirrels of both species rotate and carve the nuts to create the grooves. These new videos also show that squirrels can fit the nuts between twigs by carving the nuts. We think that these direct observations clearly support our claim, but agree that it was oversight not to included them in the first draft. See Videos 6-8.

2) Related to the point above, all speculation about the evolution of this behaviour in flying squirrels and the potential underlying cognitive mechanisms should be removed from the article, since the data are simply too preliminary at present to address these points.

The description about probable evolution and cognitive mechanisms of this behavior are removed or touched on only lightly in the context of discussion wrt to the grooving behavior itself.

3) If possible, the authors should consider a more robust analysis of nut retention in the trees, with and without grooves, or a comparison of how nuts fare on the ground versus in the trees, to support their arguments (see also comments by Reviewer #3).

As stated clearly in the text now, we did not find nuts stored on plants without grooves. Comparison of how the nuts fare on the ground versus stored on the trees is an interesting topic, which could be a very useful research project for the future. I added this possibility in the text.

There is also scope for polishing the presentation, and we recommend a thorough language check.

We have checked the whole text for language.

Finally, please note that eLife has adopted the STRANGE framework, to help improve reporting standards and reproducibility in animal behaviour research. In your revision, please consider scope for sampling biases and potential limitations to the generalisability of your findings:

https://reviewer.elifesciences.org/author-guide/journal-policies

https://doi.org/10.1038/d41586-020-01751-5

Thanks! We have checked and revised to whole paper to improve the reproducibility of this study.

Reviewer #1 (Recommendations for the authors):

I really enjoyed reading this paper and I commend the authors on the great detail with which they have recorded the behaviour. I have a few suggestions to make that I think would help to improve clarity and support to some of the text as it currently stands.

Thanks!

line 72-73: this sentence does not make sense, please rephrase.

It has been revised make it clear.

line 96-97: expand upon what "actively associated" means here, i.e., just briefly state some of the behaviours observed since this is key.

You never see the squirrels actually making the grooves but it is assumed, so I think you need to clearly state this to the reader.

Can you tell us how many unique individuals were seen in your videos? at least a minimum number?

We stated some typical behaviors about the squirrels here.

Three new videos about the carving behaviors are added to the text, which shows the squirrels were actually making the grooves.

Totally 47 times of squirrels’ activities, including nuts carving, fixing, passing and take-off were seen in our videos.

line 129: after forest, do you have a reference you can cite?

We have not published the canopy width data, but by field observation. Therefore, no reference could be cited here. I added some estimated values and revised the words.

line 163: we hypothesize, rather than guess… this is also an example of a sentence needing rephrasing due to grammar issues (see last point).

Revised as suggested.

line 165: I think you could clarify what learning ability you are talking about. At the moment this sentence is confusing because it seems like you are stating this is potentially tool use? It would be good to be more clear what you mean here and perhaps to also clearly state that this isn't tool use, but again to focus on or better explain what aspects of clever cognition this behaviour is evidence of.

The whole paragraph is deleted and then contents about tool use would not be discussed in the paper.

line 168: why animal cranial zootomy? In general I could not follow your thinking in this sentence.

We removed these words.

line 196: reference for sentence ending with 88%?

Added as suggested.

line 224: what do you mean by dominant trees?

Revised. It is referred to trees with DBH>=20cm.

Figure 2 caption: A) one groove (?) – the word groove is missing I think.

Yes. Revised as suggested.

Figure 3: although I find the angles very interesting I think the text fails to make clear why the angle might matter. Could you include in the text for example a clear explanation of why twig angles 25-40 degrees are most frequent for this behaviour?

In L99-104, there is an explanation for why twig angles 25-40 degrees matter.

Figure 4 caption: I think maybe missing some words between lines 403 and 404 or else not clear

Is it “nuts”? It is added.

Reviewer #2 (Recommendations for the authors):

A bit more development of the evidence for cache loss through decomposition ('hoard rot' as it is sometimes referred) or germination and the environmental correlates of that would be helpful earlier on for the reader to understand the observations you present.

We’ve added one sentence to deal with this suggestion. Furthermore, your perceptive comment has led us to the understanding that some very interesting experiments are possible to better understand the significance of this phenomenon. We hope to conduct them soon.

Overall the paper would benefit from some copy editing for clarity, as some sentences are a bit difficult to follow in their current form.

With sentences and words revised one by one.

I have two concerns about attributing the caching to the squirrels that could be resolved by the following:

1. Direct observation of the squirrels performing this behavior.

2. Incisor matching – do the grooves carved out of the nuts match the width of the typical width of the two incisors of these species?

3. Consideration of the following: why would an arboreal species, such as a flying squirrel, cache nuts only on the lower branches of these plants, rather than higher up? That these behaviors only appear to be at eye-level to humans does make me question why squirrels would limit themselves to this height as well.

As above, we attach three new videos to show that squirrels of both species rotate and carve the nuts to create the grooves. The new videos also show that squirrels may re-fix the nuts between the twigs by further carving the nuts. These direct observations support our claims of caching innovation better.

Wrt to incisor matching, we also added new description in the Discussion to strengthen interpretation of the grooving behavior. See L113-114.

Line specific suggestions:

Line 25 – the use of 'evolutionary challenge' – I would argue it is more an ecological challenge.

Revised as suggested.

Line 29 – specify what nation/region Hainan Island is in.

Revised.

Line 33 – the mortise and tenon joint has a long history outside China as well; it may be more recognizable to readers to briefly articulate what the joint is used for/what it looks like rather than specify a nationality.

Good point. Revised accordingly.

Line 52 – not clear what species of nuts are being discussed here, so the comment that they are in clusters doesn't immediately register. Clustering is perhaps a more species-specific phenomenon (most of the nuts I am familiar with in a temperate zone do not cluster), so this requires more explanation here.

The nut clusters are not emphasized here. We now state only that squirrels can hang some fruits on the branches. We have revised the whole sentence to meet problems identified in by the reviewers, and hope that things will be clearer now.

Line 56 – again, not sure this is necessarily an evolutionary challenge but an ecological one.

We agree that ‘ecological’ is better here and have revised accordingly.

Line 58 – Write out examples here. The Hunt et al. paper doesn't seem applicable here as it focuses on learning in locomotion, rather than cognitive food handling decisions. I would also avoid the use of the term 'smart' in discussing animal cognition; it is to me more of an anthropomorphic term and isn't necessary to describe the behaviour observed here, but I acknowledge this may be more of a personal preference.

These two sentences have been removed from the text and, as above, we understand that our initial thoughts about “smart” decision-making behavior were premature. Thus, we’ve restructured the paper so that any arguments about cognitive behavior are hypotheses developed from the data that we present.

Line 54 – 'most' nuts – would like to see some data to back this up, or at least examples of species that are commonly harvested by squirrels and therefore relevant here.

We have revised the paragraph to make the context smooth.

Line 59 – is there evidence that squirrels use tools elsewhere?

We removed the tool use description here.

The second paragraph was recognized with the first one to make the context smooth.

Line 68 – I would modify this – the behavior hasn't been reported elsewhere (have you checked databases of nature observations to be sure?)

Revised as suggested. We have checked the literature and do not find previous reports of this behavior.

Yes, this is true, and these references have been added to improve the completeness of the story. Thank you!

Line 69 – Perhaps not to facilitate the physical lodging of a nut into a crevice, but other species of squirrels do indeed handle nuts prior to caching which improves the success rate of storage (see for example Fox 1982, Steele et al. 2001, Xiao et al. 2010).

With sentences revised as suggested and new references cited. Line 79.

Supplementary Figure 1 would be helpful to have in the main text.

Removed to the main text as Figure 1. And all figure numbers are changed correspondingly.

Figure 1 – I think providing a visual comparison to the type of joint you are likening these carvings to would be helpful here, because these nut carvings appear quite different than what I would have expected given what my understanding of the mortise-tenon joint is.

Mortise-tenon joint is a convex-concave structure. We liken the twigs fixed the nuts as “tenon” which is convex structure, and the grooves on the nuts as “mortise” which formed a concave structure into nuts to receive a tenon. These sentences are added to the text. L32-34,138-139.

Line 132 – what is the criterion for "fresh"? – OK, I see now in the methods further down.

Yes, it is described in the methods.

Line 133 – how were nuts marked in the first survey to enable these conclusions?

We tagged all the plants using plastic tags. This is now mentioned in the revised methods. L252.

Paragraph at Line 163 – this is very speculative; which given that this is more of a natural history observation, is fine, but it could be much more developed in order to put this study into context and to demonstrate its value. Evolution of learned behaviors that enhance survival and reproduction is a rich topic in animal behavior, and evolution of food caching behaviors in particular has received significant attention. Feeding innovations and their relation to behavioral evolution are important (as you note, tool use in corvids and parrots are excellent examples), and perhaps describing these observations against that background with citations to the literature would help make sense of this story.

We removed the whole paragraph for the speculative contents.

Line 170 – This is late in the paper to introduce the fact that there are nine species in this region – this should be moved up.

Moved up as suggested.

Line 173 – have they evolved this behavior? Or is it learned or innovated? Evolution is a bold claim in the absence of familial data including fitness metrics and given how tightly clustered in time and space these observations are.

With sentences revised.

Line 180 – this is also a 'just-so' fallacy; you observed the persistence of these nuts over a very short time and did not present controls of what happens to these same species of nuts should they be stored on the ground or in the leaf litter. My experience in the tropical rain forest leads me to believe that this behavior is highly likely to have that effect of cache protection, but just because it makes logical sense does not make it data.

As suggested, we now mention that the protective value of the hanging-nut caches merits further study with comparison to the fates of nuts on the ground.

Reviewer #3 (Recommendations for the authors):

The authors described and documented a nut-storing behaviour of two species of flying squirrels that is not previously mentioned in the literature. The authors found that the two species of flying squirrels made grooves in nuts so that they can be fixed between twigs of different species of plants and shrubs. The authors conclude that the squirrels of these populations are evolved or learn to do so, as well as placed such behaviour in tool-use context and claim that they are 'smart'.

Overall, I enjoyed reading the manuscript, and knowing more about squirrels' behaviour. I also find the work is important, because there isn't proper documents stating this nut grooving behaviour in squirrels. This will spark an interest of understanding the causes and consequences of this nut grooving behaviour, which advances different fields such as animal behaviour and cognition, behaviour ecology, evolution and ecology.

Thanks!

Despite the strengths of the study, I do have several suggestions to improve the manuscript:

1) One of the goals of the study is to show that these squirrels are smart. Now I am not saying that they are not, but we need proper evidence, systematic investigations, and analyses to establish this claim. For now, these claims are not entirely supported by analyses or proper measurements; the definition of 'smart' and relating nut grooving behaviours to tool use is not complete. For example, no evidence indicating this behaviour involves complex cognition or cognitive process – smart, or it is related to fast learning performance – smart. All we know is the squirrels did it, but how they acquire such skills and whether it involves in complex or advance cognition is another story. With this in mind, many wordings appear to lead readers think that this is established, which is not the case.

Yes, you are right. Our surprise and enthusiasm about discovering that these squirrels do this have likely pushed us to assume too much. Thus, we have revised the text and title to focus on the behaviors of grooving and storing nuts, and to downplay conclusions relating to cognitive or cognitive process. Once we have more evidence to show how squirrels develop such skills, and whether the behaviour involves complex or advance cognition, or fast learning performance, we will write another story.

2) Analyses – the current analyses basically is 0. Can't the authors pay a little more efforts in analyses, say using (M)ANOVA and post hoc tests to examine whether the number of as well as the depth of grooves differ in nuts that store on ground and (live and dead) trees? Or depending on the shape of a nut too? More importantly, out of how much footage did the authors capture the flying squirrels did the nut grooving behaviours? what other animal species interacted with those nuts? These are the central aims of the manuscript but are not done properly.

We have added detailed descriptions about how histograms and t-tests were applied.

A total of 47 footage clips capture flying squirrels, as is now mentioned in the first paragraph of results and discussion.

Interestingly, other larger-bodied squirrels passed by some plants with a stored nut, but they did not touch the nuts. Some mice were observed trying to take stored nuts, as is now mentioned in the text. L152.

3) The current presentation of the manuscript is largely oriented to plants, and less about animal behaviour (definitely not involved in animal cognition). This appears to be a bit contradictory to what the authors would like to present or what they found interesting; that the grooving behaviour of seeds in flying squirrels is novel. Suggest thinking about how to reorganise this information and focus on animal behaviour

We have rephrased the text to amplify the new finding of the grooving behaviors, which has been previously unrecognized in squirrels. We have revised the text to focus on the special behavior and how it changes nuts themselves, but also describe the vegetation sites associated with the suspended nuts that we discovered.

4) Figures – please present proportion or percentage on y axis, place the actual value on top of each bar in the bar graph; this will make information more presentable as well as increase the transparency of data.

All figures are revised as suggested.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The article has been much improved but there are some remaining issues that need to be addressed, as outlined below:

1) We appreciated the addition of the new videos to demonstrate the squirrels' active interaction with the nuts, but could you please address Reviewer #3's alternative explanation directly in the text and provide any further clarification regarding the carving or cracking behaviour?

We’ve added one new Video 9 that provides direct evidence and example of the process used by squirrels for fixing nuts. The video documents the behaviour of a squirrel that was holding a nut in its mouth and engaged in the process.

I think that the word ‘cracking’ does not appropriately express what was done by squirrels. We feel that use of the word ‘cracked’ would be misleading as the grooves are strictly surficial. This seems to be important as we point out below (Lines 128-130) “Nonetheless, none of the chewed grooves that we observed damaged the endosperm of the nut, and thus the squirrels seemed to minimize the potential impacts of fungi during storage.” The grooves, in our opinions are in fact “carved” carefully into the surface without breaking through to the inside of the nut. Thus, we have followed our antecedent expression of “chewing” in lines 85-88, taking the opportunity to establish that we infer that the chewing leads to “carved” surficial grooves. Clearly, when one refers to “cracking” nuts, say during the holiday season, it means to break the outside casing of the nut in order to get to the inside of the nut. Thus, we fear the use of “cracking” could be easily misleading. In an attempt to avoid any confusion, we have replaced “carving” with “chewing” until our word use had been clearly defined.

2) Similarly, please modify the text to be explicit about which parts of the behaviour you have direct video evidence for and which actions are inferred and, therefore, still require further supporting evidence. This will improve transparency and help readers evaluate your findings.

We added vdeo number after each action to refer the readers to direct video evidence for our claims in Line 88-90. These actions include chewing to carve grooves (Video 6-7), as well as fixing (Video 9) and removing nuts (Video 2, 3, 5) and visiting a storage site (Video 1, 4, 8). The sentence was also revised to clearly state that the videos provide direct evidence to support aspects of nut storage behaviour employed by these two squirrel species.

Reviewer #3 (Recommendations for the authors):

Thank you very much for inviting me to review this paper again. I also thank the authors for making a great effort to revise the paper, and most of my previous suggestions/concerns have been taken into the revision. The revised paper is still interesting as it documents the grooved nut in between twigs that may be made by this species of squirrel (a behaviour that is not fully understood).

In the past version of the manuscript, one of the main weaknesses of the paper was the absence of evidence showing squirrels made grooves and fixing them in between the twigs. In this version, the authors submitted three new videos (videos 6-8), and they are useful for seeing this species of squirrel handling a nut. However, videos 6 and 7 are showing a squirrel rolling a nut and attempting to 'carve' them and Video 8 is a squirrel finding a nut and attempting to take it out between the twigs. The terminology 'carving' is debatable, as to some squirrel experts, this appears to be 'cracking' the nut shell instead of 'carving'. None of these videos really shows direct evidence as in a squirrel grooving a nut and fixing it in between the twigs. If the authors insist that the squirrels do so (which from line 92 appears to be the case), we would need videos that show a squirrel making these grooves and fitting the nut in between twigs.

As above, we’ve added a new Video 9 that provides direct evidence of the process that squirrels use to fix the nuts.

After carefully considering the differences between these two words, “carving” has been mostly replaced by “chewing” throughout the paper. See above.

https://doi.org/10.7554/eLife.84967.sa2

Article and author information

Author details

  1. Han Xu

    1. Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China
    2. College of Forestry, Hainan University, Haikou, China
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing
    Contributed equally with
    Lian Xia and John R Spence
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1085-3344
  2. Lian Xia

    College of Forestry, Hainan University, Haikou, China
    Contribution
    Formal analysis, Investigation, Writing – original draft
    Contributed equally with
    Han Xu and John R Spence
    Competing interests
    No competing interests declared
  3. John R Spence

    Department of Renewable Resources, University of Alberta, Edmonton, Canada
    Contribution
    Writing – original draft, Writing – review and editing
    Contributed equally with
    Han Xu and Lian Xia
    Competing interests
    No competing interests declared
  4. Mingxian Lin

    Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China
    Contribution
    Investigation, Writing – original draft
    Competing interests
    No competing interests declared
  5. Chunyang Lu

    1. Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China
    2. College of Forestry, Hainan University, Haikou, China
    Contribution
    Investigation, Writing – original draft
    Competing interests
    No competing interests declared
  6. Yanpeng Li

    Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China
    Contribution
    Investigation, Methodology, Writing – original draft
    Competing interests
    No competing interests declared
  7. Jie Chen

    Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China
    Contribution
    Writing – original draft
    Competing interests
    No competing interests declared
  8. Tushou Luo

    Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China
    Contribution
    Investigation, Writing – original draft
    Competing interests
    No competing interests declared
  9. Yide Li

    Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China
    Contribution
    Data curation, Writing – original draft
    Competing interests
    No competing interests declared
  10. Suqin Fang

    State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Methodology, Writing – original draft, Writing – review and editing
    For correspondence
    fangsuq5@mail.sysu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1324-4640

Funding

Science and Technology Basic Work Project from Ministry of Science and Technology of the People's Republic of China (2019FY101607)

  • Han Xu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Wenhao Qin, Chuanwen Yu, Fenglin Huang, and Tao Zhang for helping to collect the field data. We also appreciate suggestions from Yi Ding from Chinese Academy of Forestry that improved the manuscript, and assistance from Fang Liu from the Chinese Academy of Forestry, and Qiang Zhang from the Institute of Zoology, Guangdong Academy of Sciences in identifying the animals. Science and Technology Basic Work project from Ministry of Science and Technology of the People’s Republic of China (2019FY101607).

Senior Editor

  1. Christian Rutz, University of St Andrews, United Kingdom

Reviewing Editor

  1. Ammie K Kalan, University of Victoria, Canada

Reviewers

  1. Ammie K Kalan, University of Victoria, Canada
  2. Pizza Chow, University of Chester, United Kingdom

Version history

  1. Received: November 16, 2022
  2. Preprint posted: November 21, 2022 (view preprint)
  3. Accepted: May 26, 2023
  4. Accepted Manuscript published: June 13, 2023 (version 1)
  5. Version of Record published: July 7, 2023 (version 2)

Copyright

© 2023, Xu, Xia, Spence et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Han Xu
  2. Lian Xia
  3. John R Spence
  4. Mingxian Lin
  5. Chunyang Lu
  6. Yanpeng Li
  7. Jie Chen
  8. Tushou Luo
  9. Yide Li
  10. Suqin Fang
(2023)
Flying squirrels use a mortise-tenon structure to fix nuts on understory twigs
eLife 12:e84967.
https://doi.org/10.7554/eLife.84967

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https://doi.org/10.7554/eLife.84967

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