# Jumping out of trouble: Evidence for a cognitive map in guppies (Poecilia reticulata)

Hannah de Waele, Catarina Vila-Pouca, Dimphy van Boerdonk, Ewoud

Luiten, Lisanne Leenheer, David Mitchell, Regina Vega-Trejo, Alexander

Kotrschal

► To cite this version:

Hannah de Waele, Catarina Vila-Pouca, Dimphy van Boerdonk, Ewoud Luiten, Lisanne Leenheer, et al.. Jumping out of trouble: Evidence for a cognitive map in guppies (Poecilia reticulata). 2022. [hal-04709513](https://hal.science/hal-04709513v1)

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https://hal.science/hal-04709513v1

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<figure><img src="image_1.png" /></figure>
1 Jumping out of trouble: Evidence for a cognitive map in guppies (*Poecilia reticulata*)

2 Hannah de Waele¹*, Catarina Vila Pouca¹, Dimphy van Boerdonk¹, Ewoud Luiten¹, Lisanne M.

3 Leenheer¹, David Mitchell², Regina Vega-Trejo²,³, Alexander Kotrschal¹

4 Hannah De Waele and Catarina Vila Pouca should be considered joint first author.

5

6 ¹Department of Animal Sciences: Behavioural Ecology, Wageningen University and Research, 6708

7 WD Wageningen, The Netherlands

8 ²Department of Zoology, Stockholm University, Svante Arrhenius väg 18B, 10691, Stockholm,

9 Sweden

10 ³Department of Zoology, Edward Grey Institute, University of Oxford, Oxford OX1 3SZ, UK

11

12 *Corresponding author:

13 Hannah De Waele

14 Behavioural Ecology Group, De Elst 1, 6708 WD, Wageningen, The Netherlands

15 +32 493 33 19 09

16 Hannah.dewaele@wur.nl

17

18

19

20

21 Word count: 7.633

22 Number of tables: 0

23 Number of figures: 6

24
25 Abstract

26 Spatial cognitive abilities allow individuals to remember the location of food patches, predator hide-
27 outs, or shelters. Animals typically incorporate learnt spatial information or use external
28 environmental cues to navigate their surroundings. A spectacular example of how some fishes move is
29 through aerial jumping. For instance, fish that are trapped within isolated pools, cut off from the main
30 body of water during dry periods, may jump over obstacles and direct their jumps to return to safe
31 locations. However, what information such re-orientation behaviour during jumping is based on
32 remains enigmatic. Here we combine a lab and field experiment to test if guppies (*Poecilia reticulata*)
33 incorporate learnt spatial information and external environmental cues (visual and auditory) to
34 determine where to jump. In a spatial memory assay we found that guppies were more likely to jump
35 towards deeper areas, hence incorporating past spatial information to jump to safety. In a matched vs.
36 mismatched spatial cue experiment in the field, we found that animals only showed directed jumping
37 when visual and auditory cues matched. We show that in unfamiliar entrapments guppies direct their
38 jumps by combining visual and auditory cues, while in familiar entrapments they use a cognitive map.
39 We hence conclude that jumping behaviour is a goal-directed behaviour, guided by different sources
40 of information and involving important spatial cognitive skills.

41 **Key words:** orientation, spatial cognition, mental map, aerial jumping, guppy
## 42 Introduction

Mobile animals need to navigate to locate resources, find mates, or return to their home area, while simultaneously minimizing predation risk (Braithwaite & Girvan, 2003; Noda, Gushima, & Kakuda, 1994). Animals rely on a range of spatial cognitive skills to perform these tasks; they can recognize complex cues, form long-term memories, and regain their bearings after disorientation. For example, Clark's nutcrackers (*Nucifraga columbiana*) can memorize the locations of thousands of caches up to nine months after caching (Balda & Kamil, 1992), and displaced honeybees find their hive by using spatial features learned during a single flight (Degen et al., 2016). Some fish species also have excellent spatial learning and memory abilities (Teyke, 1989; Warburton, 2003). Atlantic salmon (*Salmo salar*), goldfish (*Carassius auratus*), fifteen-spined sticklebacks (*Spinachia spinachia*), and corkwing wrasse (*Crenilabrus melops*) can locate a food reward using visual landmarks (Braithwaite, Armstrong, McAdam, & Huntingford, 1996; Hughes & Blight, 2000; López, Broglio, Rodríguez, Thinus-Blanc, & Salas, 1999; Salas et al., 1996), even when released in an unfamiliar spot (Rodriguez, Duran, Vargas, Torres, & Salas, 1994). There is also evidence that some fishes (e.g. Mexican cave fish (*Astyanax mexicanus*), goldfish (*Carassius auratus*), and butterflyfishes (family Chaetodontidae) use cognitive maps to navigate their surroundings (Burt De Perera, 2004; Reese, 1989; Rodriguez et al., 1994). Cognitive maps are internal map-like representations of relationships between different landmarks in their environment, created through spatial learning (Gould, 1986; Toates, 1980; Tolman, 1948). Such an internal representation of the environment allows animals to adopt novel routes from previously unvisited points, removing the requirement of learning specific routes (Mazmanian & Roberts, 1983; Morris, 1981; O'Keefe & Conway, 1978; Suzuki, Augerinos, & Black, 1980).

To further understand mechanisms of spatial orientation, researchers have taken advantage of aerial jumping behaviour in fishes since it presents a clear directional choice of movement (Buo et al., 2020a). Aronson (1951, 1971) and White & Brown (2014) used this behaviour to demonstrate that gobies have a cognitive map of their habitat as they can leap from one tidal pool to another even when they have no visual contact before leaving the water. Guppies (*Poecilia reticulata*) also show aerial jumping (Soares & Bierman, 2013) and may therefore possess similar spatial abilities. In Trinidadian
69 rivers, the guppies' native range, seasonal changes and weather events connect and disconnect pools;
70 connecting them at high water and isolating them from the main river as the water recedes. By being
71 able to jump out of such pools, guppies should be able to return to the main river, escaping isolation
72 and potential death as the pools desiccate. Soares & Bierman (2013) documented guppies generally
73 starting a jump with a slow and preparatory backwards movement, followed by a forward thrust,
74 propelling the fish into the air. These small fish are able to jump approximately 3.5 times their body
75 length in height, with a positive correlation between jump height and back-up distance (Soares &
76 Bierman, 2013). This suggests that guppies can actively alter jump height and likely also distance.
77 Additionally, the preparatory phase suggests jumping is not purely reflexive, but involves a clear
78 motive. However, whether guppies are able to direct their jump towards a specific location, and what
79 information they base their jumping direction on, remains untested.

80 As guppies are capable of using spatial information in maze-experiments (Burns & Rodd, 2008b;
81 Chapman, Ward, & Krause, 2008; Lucon-Xiccato & Bisazza, 2017b, 2017a; Miletto Petrazzini,
82 Bisazza, Agrillo, & Lucon-Xiccato, 2017), we tested if and how guppies base their jumping directions
83 on spatial information in two experiments. In the laboratory, we tested whether guppies use the
84 memory of specific landmarks (cognitive maps) to jump out of 'trouble' in a known small water body.
85 We used water level changes in combination with entrapment in desiccating pools to test if guppies
86 remember which areas are safe to jump to. In this relatively small arena, the shallow areas become dry
87 land and the deep areas remain water-filled pools. We predicted that if guppies memorize their
88 environment and incorporate that spatial information, they should aim their jump towards the pools.

89 In a second experiment in the wild, we tested whether guppies show directed jumping behaviour when
90 entrapped in unknown small water bodies. Such a situation may occur during flash floods when a fish
91 gets washed ashore and finds itself in a desiccating body of water that lies outside its known area. We
92 hypothesized that, in the scenario where the fish has no previous information about the specific
93 environment, they should use general landmarks like visual and/or auditory cues. Hence, this scenario
94 excludes the possibility of basing jumping direction on cognitive maps, and requires them to rely on
95 external, general landmarks. For instance, the open canopy may function as a general landmark as it
can indicate the middle of the river. Sunlight and gap size in the tropical forest determine different light environments, with canopy openness decreasing from the centre of the stream to the riverbanks (Endler, 1992). The canopy structure has various visual aspects that the guppies could use for orientation, including the increased light level in the middle of the river. There is evidence that several fish species show the ability to orient themselves towards light sources (phototaxis) (Barki, Zion, Shapira, & Karplus, 2014; Goodyear & Ferguson, 1969). In addition to phototaxis, the trees at either side of the river can function as a general visual landmark on which fish orient to find the middle of the stream. Although the canopy is distant, fishes have been shown to use external, general cues outside of the water to orient themselves (Rodriguez et al., 1994). Hence, the various visual aspects of the canopy structure can function as a general visual landmark on which fish orient to find the middle of the stream. Another general landmark may be the sound of flowing water as a loud local riffle may indicate flowing water and therefore a safe jumping direction. Sound can be important for orientation in aquatic animals because of its highly directional nature (Hawkins & Popper, 2018). For example, sound is a meaningful orientation and attraction cue for larvae of reef fishes (Montgomery, Jeffs, Simpson, Meekan, & Tindle, 2006; Simpson, Meekan, Montgomery, McCauley, & Jeffs, 2005; Tolimieri, Jeffs, & Montgomery, 2000). Since the sound of a riffle indicates a 'safe' direction towards water, it may trigger the guppies to jump towards that direction. We investigated their jump direction in a cue-conflict/cue-matching experiment, where we exposed guppies to different spatial combinations of riffle sounds and canopy openness. This experiment tested which of two general landmarks, sound or canopy openness, guppies base their jumping decision on. We hypothesized that both cues are important and predicted that guppies will show the highest directional preference when both cues match.
118 **Materials and Methods**

119 We aimed to test the hypotheses that guppies can (1) use a cognitive map based on learnt spatial information, and (2) can incorporate external environmental cues (visual and auditory) to determine where to safely jump. To do so, we ran two experiments.

122 **Experiment I - Testing for a cognitive map in the laboratory**

123 In a laboratory experiment, we aimed to understand whether guppies use cognitive maps to direct their jumps. The study was carried out in the animal facilities at Wageningen University and Research in April 2020. Guppies used in the experiment were 20 adult females; laboratory-reared descendants of fish collected from a high-predation population from the Aripo river in Trinidad. Prior to experiments, fish were housed in same-sex groups of six in 4 litre flow-through tanks at Wageningen University.
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127 Temperature was kept at 24°C with a 12:12h dark/light cycle. Fish were fed twice daily on seven days a week with flake food and freshly hatched *Artemia salina* nauplii.
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130 Two identical experimental tanks (70 x 35 x 40 cm) were filled with gravel and shaped into four quadrants of different depths (two shallow areas, two deep areas; Figure 1). Two-centimetre ridges separated the quadrants from each other. Additionally, two markers, consisting of wooden sticks, were used to mark the division between the shallow and deep areas and to serve as a visual landmark for the fish. The water level in the experimental tank could be altered by siphoning out water or adding water through two identical tubes attached to the sides of the tank. The shallow areas were approximately five cm deep at high-water. At low water level, the shallow areas fell dry. The deeper areas consisted of a slope, starting its decline at the border with the shallow areas. During high water the deep area ranged from 5 to 17 cm in depth. During low water, the deepest area was 12 cm deep. In the middle of the tank there was a circular testing arena with a diameter of 9 cm that was surrounded by a 1.5 cm high ring of gravel so that the water level of the rest of the tank was not visible from the inside the testing arena during low water levels. The circular testing arena had a depth of approximately 3 cm at low levels, imitating an isolated tidal pool. A camera was mounted above the experimental tank. Both deep areas were equipped with a filter during the habituation period of the test, and stones were used in the corners of the shallow areas to limit access to the tubes used to alter the water level.
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<figure><img src="image_2.png" /><figcaption>**Figure 1.** Set up of the experimental tank: view from above (A), and view from the side at low water (B) with depth representation. The experimental tank was filled with gravel to create four quadrants of different depths (two shallow areas, two deep areas). Two-cm ridges separated the quadrants from each other. Different shades of blue represent different depths, with darker tones marking the deeper areas. The “T” marks the testing arena.</figcaption></figure>

Ten females were simultaneously transferred into one of the experimental tanks at high water and allowed to habituate for one hour. This and all subsequent procedures were done in parallel in the two experimental tanks. The fish were exposed to the experimental tanks for three consecutive days. They then experienced three 2-hour periodical flood cycles per day. Within one cycle, the water receded for approximately 25 min. After a 10 min rest period, the water started rising for 25 min. This was repeated twice per cycle. After finishing the third cycle the fish were fed. The same procedure was repeated on the second day. On day 3, after one full cycle, the water level was lowered once more, and the guppies were caught and moved to temporary holding tanks. Guppies were caught individually with a net, then transferred into the circular testing arena with a cup, and observed for their jumping behaviour. If a fish did not jump within 10 min, they were removed from the testing arena and returned to their holding tank and excluded for further testing. Latency to jump and landing location were determined from video footage. Note that before the testing phase the shallow areas fell completely dry at low water, yet, during the testing phase, a depth of 1 cm was maintained to avoid fish jumping on dry land and possibly injuring themselves. Thus, even when they jumped towards the shallow areas, they landed in shallow water. The water level was well below the visible threshold from within the testing arena.
167 **Experiment II - Testing for general spatial cues in the wild**

168 In a field experiment, we aimed to understand whether guppies use auditory cues of moving water
169 and/or visual cues of the open canopy to orient themselves when jumping. To do so, we conducted an
170 experiment in the lower Aripo river in Trinidad (Lat.: 10.6667; Long.: -61.2285) in March 2020. This
171 site was chosen due to its steady guppy population, its pronounced network of riffles and pools, and a
172 relatively high canopy openness in the middle of the river. A riffle was defined as a small waterfall or
173 a localized fast-flowing current generated by numerous boulders within the river (Figure S2). Apart
174 from the sound of the riffles, the site was calm and the riffles were spaced apart, limiting the sound
175 interference between different riffles.

176 We selected three locations towards the left bank and two locations towards the right bank so as to
177 vary the direction to the open canopy. At each of these locations, two different spatial arrangements
178 (treatments) relative to two cues were created on either side of the riffle (Figure 2). The riffle was
179 either spatially matching – at the same side of the open canopy – or contrasting – at the opposite side
180 of the open canopy (Figure 2). Water depth ranged from a few centimetres close to a riffle to 1 m in
181 the deeper areas. Note that all locations were within the stream for ethical reasons. Thus, all jumping
182 directions led back to water.

183 Nine trials were done at two locations, and eight trials were done at the other eight locations. Female,
184 male, and juvenile guppies (N = 492) used in this experiment were collected with dip and butterfly
185 nets within a range of twenty meters around the test site in pools and the main stream. We chose to
186 assay females, males, and juveniles to obtain a more representative sample of jumping behaviour in
187 the species, but made no specific predictions on how females, males, and juveniles should differ in
188 their jumping behaviour.
<figure><img src="image_3.png" /></figure>

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**Figure 2.** Schematic overview of the study site. Test locations are marked A through E. At each location both treatments (M: matching cues; C: contrasting cues) were tested.

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During jumping trials, guppies were placed in opaque white Styrofoam cups (5.5 cm diameter, 3.5 cm height) that were filled with 30 ml of fresh stream water so that the height difference between the water surface and cup edge was roughly 1 cm. At the start of a trial, six cups were placed near the edge of a boulder at the riffle side and guppies were individually placed into the cups. Trials were filmed for 10 minutes using an overhead stationary GoPro camera, mounted on a PVC tube structure (Figure S2). Observers kept a distance of at least one meter.

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For each trial, individual fish were scored for whether or not they jumped within 10-minutes and the jump latency was recorded. Subsequently, for the fish that jumped (n = 275), still frames were isolated from the video footage and analysed to estimate the jumping angle (Fig. S1). A first frame was selected when the fish started to protrude above the water surface. In this frame, a line was drawn from the fish's centre to the riffle centre. This line was used as a reference and was set at 0 degrees using modular arithmetic. Thus, all subsequent calculated angles were relative to this line. A second frame was then selected where the fish crossed the edge of the cup. In this frame, a line was drawn from the fish's centre when starting the protrude to the fish's centre when crossing the edge of the cup. The
angle between the two drawn lines was calculated by measuring the shortest clockwise path using the Protractor feature in the PicPick software (version 5.0.7; 2020). This angle is referred to as the jumping angle. Three jumps were excluded from the analysis since the angle was impossible to measure due to low quality footage. The jumping angle was calculated in 272 individual guppies by a single researcher to avoid observer differences.

## Statistical analysis

### Experiment I:

To test whether the jumping rate towards the pools was significantly different than random chance we used a two-sided binomial exact test. Subsequently, to test whether the latency to jump varied between the fish jumping towards deep areas or the fish jumping towards the shallow areas we used a Wilcoxon rank sum test.

### Experiment II:

To test for differences in Jumping frequency, we used a linear mixed model (binomial distribution) with Treatment (opposite or matching cues), Life-stage (male, female, juvenile) and the interaction of Treatment x Life-stage as predictor variables, as well as a random factor for Location. We used the 'lme4' package (Bates, Mächler, Bolker, & Walker, 2014). Model terms were tested for significance using the ANOVA function in the car package (Fox & Weisberg, 2018), specifying Type III Wald chi-square tests. If Life-stage was a significant predictor in the model, we assessed Tukey corrected multiple comparisons between Life-stage levels using the 'lsmeans' function in the 'emmeans' package (Lenth, Singmann, Love, Buerkner, & Herve, 2019). The interaction between Treatment and Life-stage in this model was removed from the model as it was marginally non-significant and uninformative for our research question (p = 0.0506).

To test for differences in Jump latency, non-jumpers were excluded. Subsequently, we used a linear mixed model (normal distribution) with Treatment, Life-stage, and the interaction of Treatment x Life-stage as predictor variables, as well as a random factor for Location using again the 'lme4' package. Model terms were tested for significance using the ANOVA function in the car package (Fox &
232 Weisberg, 2018), specifying Type III Wald chi-square tests. The interaction between Life-stage and 233 Treatment in this model was removed from the model as it was non-significant and uninformative for 234 our research question (p = 0.403). If Life-stage was a significant predictor in the model, we assessed 235 Tukey corrected multiple comparisons between Life-stage levels using the ‘lsmeans’ function in the 236 ‘emmeans’ package (Lenth et al., 2019). To meet the homogeneity and normality assumptions, we 237 performed a log (10) transformation on Jump latency.

238 To investigate the role of sound and/or canopy cover for jumping orientation, we used circular 239 statistics techniques. The Rayleigh test was used to assess the uniformity of the jumping direction. If 240 no clear direction is preferred, then all directions would be approximately equally covered on the 241 circumference of a circle, visible in a circular uniform distribution. Any deviation from a circular 242 uniform distribution would be indicative of a preferred direction. The Rayleigh test assumes that the 243 data follow a unimodal and symmetrical von Mises distribution (Landler, Ruxton, & Malkemper, 244 2019). To rule out multimodal departures and unknown symmetry, the Hermans-Rasson test was 245 performed. All statistical analysis were performed in R-3.6.2 (r Development Core Team 2012).
246 **Results**

247 **Experiment I:**

248 In the laboratory experiment testing if guppies direct their jumps using a cognitive map, all female guppies jumped out of the testing arena. Of the twenty tested animals, 16 jumped towards a deep area and 4 jumped towards a shallow area (Figure 3A). Thus, fish were more likely to jump towards the deeper pools than expected at random with a 50:50 chance (N = 20; Z = 16; p = 0.006).

252 We found no difference in the latency to jump between the guppies jumping towards the deep areas compared to the guppies jumping towards the shallow areas (Wilcoxon rank sum test; N = 20; W = 45; p = 0.237; Figure 3B). Note that there were only 4 fish in the group jumping towards the shallow areas, thus, there is little power to assess differences in the latency to jump towards these different areas.

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Percentage of jumps (A) and latency to jump (B) for guppies jumping into the deep areas (dark blue) and onto the shallow areas (light blue). Black markers in B show individual data points.</figcaption></figure>

260 **Experiment II:**

261 In the field experiment testing for general spatial cues, 55.9% (275/492) individuals jumped out of the testing cup, while 44.1% (217/492) did not jump within the 10-minute experimental time. We found
no differences in jumping frequency between the treatment groups ($\chi^2_{1,270} = 0.019$; p = 0.892), but
jump frequency differed between juveniles, females and males ($\chi^2_{2,270} = 23.392$; p < 0.0001). Pairwise
comparisons showed that juveniles jumped significantly less than males and females (Juvenile – Male:
p = 0.019 and Female – Juvenile: p < 0.0001).

When filtering the data to only fish that jumped, we found no differences in the latency to jump of fish
from the matching cues treatment compared to those from the contrasting cues treatment ($F_{1,270} =$
3.626; p = 0.057; Figure 4A). Yet, we found that juveniles, females, and males differed in jump
latency ($F_{2,270} = 23.229$; p < 0.0001; Figure 4B). Pairwise comparisons indicated that females jumped
approximately 26 seconds faster than juveniles and 19 seconds faster than males (Female – Juvenile: p
< 0.0001 and Female – Male: p = 0.012).

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Latency to jump for the different treatments (contrasting cues and matching cues) (A) and for females, males, and juveniles (B). The proportion of jumpers for each group is noted underneath the violin plot. Horizontal lines indicate medians, boxes indicate interquartile range, and whiskers indicate all points within 1.5 times the interquartile range.</figcaption></figure>
When exposed to matching cues, the jumping direction for all fish showed a significant deviation from a uniform distribution ($R = 0.253$; $Z_{135} = 8.659$; $p = 0.0002$; Figure 5A, which indicates that fish preferentially jump towards a specific direction. We found that the mean direction of the jump was 18.75 degrees relative to the middle of the riffle, pointing towards the riffle and canopy openness. In contrast, fish exposed to contrasting cues showed a randomly distributed jumping orientation, with no clear preferred jump direction ($R = 0.058$; $Z_{137} = 0.463$; $p = 0.630$; Figure 5B).

A

Matching cues

<figure><img src="image_6.png" /></figure>

B

Contrasting cues

<figure><img src="image_7.png" /></figure>

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**Figure 5.** Jumping direction distribution for (A) matching cues (n = 136) and (B) contrasting cues (n = 139). The arrow points towards the mean angle and its length reflects the concentration of data. Black dots indicate individual data points.

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When investigating the jump direction of males, females, and juveniles separately, only females in the matching cues treatment showed a preferred direction towards the two cues, departing from a uniform direction (Rayleigh test; $R = 0.342$; $Z_{60} = 7.037$; $p < 0.0009$; Figure 6A). Although the mean jumping direction for the males and juveniles appeared qualitatively similar (Figure 6A), the jumping direction did not differ from uniformity. When exposed to contrasting cues, neither of the sexes nor juveniles showed a preferred jumping direction (Figure 6B).
A

Matching cues

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

B

Contrasting cues

<figure><img src="image_10.png" /></figure>

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**Figure 6.** Jumping distribution for the different stages (female, male, and juvenile) for matching cues

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(A) and contrasting cues (B). The arrow points towards the mean angle and its length reflects the

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concentration of data.
298 **Discussion**

299 In two complementary experiments, we found evidence that guppies direct their jumps towards a
300 specific direction and use both a cognitive map and general landmarks to orient themselves. When
301 guppies were confined in a small and shallow pool in a familiar environment, they swiftly jumped out
302 to land in deeper water bodies. In contrast, when they found themselves in unknown confined water
303 bodies, they were less likely to jump, but when they did, they oriented themselves using general
304 landmarks such as the sound of moving water and canopy openness.

305 The first aim of this study was to test whether guppies have the capacity to memorize their
306 surroundings and use this information to determine their jumping direction. We found that all animals
307 jumped out of the test arena within minutes and most of the animals jumped towards areas with deeper
308 water, supporting our prediction that guppies can use cognitive maps to direct their jumps. Spatial
309 cognition has been well studied in guppies (Burns & Rodd, 2008a; Kotrschal, Corral-Lopez, Amcoff,
310 & Kolm, 2015; Lucon-Xiccato & Bisazza, 2017c). For example, they can learn to navigate a complex
311 maze consisting of up to six consecutive T-junctions (Lucon-Xiccato & Bisazza, 2017a). Our data
312 adds cognitive maps to this portfolio of spatial abilities because, similar to fishes such as the
313 amphibious spotted rock skipper and some gobies (Aronson, 1971; Buo et al., 2020b; White & Brown,
314 2014), they reliably jump to favourable areas even when the direct line of sight is blocked. Due to the
315 way our test pool was designed, the guppies could not see the water level outside of the pool during
316 the test and could only orient using the provided visual cues and/or internally stored information.

317 External visual cues included the quadrant division markers, cues from the experimental room, and
318 differences in ceiling colour. While maze experiments show that guppies typically do not rely on
319 subtle cues when navigating (Lucon-Xiccato & Bisazza, 2017a), they could have done so in this study
320 and memorized external landmarks may be part of the cognitive map of the environment. In line with
321 our predictions, we found that guppies have the ability to create cognitive maps of their environment
322 and retrieve those to direct their jump in order to successfully return to deeper bodies of water.

323 The second experiment tested how guppies react to being trapped in small water bodies that are
324 unknown to them but lie within their known home range. In these cases, they likely had no memory of
the quality of the environment they jumped towards. We hypothesized that guppies then may rely on external visual and/or auditory cues, i.e. the canopy structure and a local riffle, to orient themselves when jumping out of an isolated area. In line with our predictions, we found that only when visual and auditory cues were aligned guppies showed clear directional jumping towards the middle of the stream, while under contrasting cues no clear directional choice was evident. Guppies seem to apply multimodal orientation as several cues need to align for effective re-orientation.

Most spatial task studies in fish have focused on the relevance of learned landmarks for orientation (e.g. Buo et al. 2020b; Odling-Smee and Braithwaite 2003). Guppies in the present study were predicted to orient spontaneously without training or reinforcement. Guppies live in large and highly changing habitats. Hence, the use of cognitive maps might not always be feasible or optimal. General landmarks, such as the geometry of the environment, should provide a better tool to orient than local landmarks when stranded in an unknown area. Indeed, the shape of the environment is crucial for reorientation in fishes, rats, and pigeons, showing that large-scale geometry of the environment is more relevant than local non-geometrical cues (Cheng, 1986; Kelly, Spetch, & Heth, 1998; Sovrano, Bisazza, & Vallortigara, 2002).

In a guppy's habitat, we hypothesized that canopy openness and riffle sound may be suitable general landmarks. Brighter light conditions can be indicative of the location of the middle of the stream, as the canopy is less dense there. This should provide information about where the water is likely the deepest. On top of light level, the canopy also provides other visual features that animals have been shown to use for orientation, including specific shapes in the canopy pattern or the overall contrast between the trees at both sides and the open sky. For instance, ants orient using the high contrast cues created by canopy patterns (Ehmer, n.d.; Hölldobler, 1980) and in fishes, depending on species, positive or negative phototaxis are widespread (Barki et al., 2014; Forward Jr, Horch, & Waterman, 1972). Additionally to optical features, we hypothesized that water murmur, or the disturbance caused by water flowing against rocks at riffles, might function as a general auditory landmark, as fishes can use sound as an orientation cue (Montgomery et al., 2006). We found that guppies showed only a preferential jumping direction when the two hypothesized landmarks, open canopy and riffle sound,
352 were aligned. We conclude that both general landmarks are important for orientation and are used
353 collectively.

354 Several non-mutually exclusive hypotheses exist to explain aerial jumping in guppies, from predator
evasion (Seghers, 1973) to means of dispersal (Soares & Bierman, 2013). Here we show that guppies
355 can use aerial jumping to escape isolation. Streams in Trinidad, the guppy's environment, are highly
dynamic with rapid water level changes, and getting trapped in a desiccating pool after a flood or a dry
356 period is likely a common event. While staying in these isolated pools would mean isolation or even
357 death, uncertainty about the surroundings may prevent the guppy from jumping into an unknown
358 environment. Costs of erroneous jumping decisions include landing on dry land with the risk of
desiccation. Even though repeated flapping on dry land can help getting back into a water body,
359 impact on the rocks may still cause physical injury. We found a clear difference on whether guppies
360 jumped or not out of confinement between our two experiments. While all animals rapidly jumped
361 from the known pools in the laboratory, almost 40% of animals did not jump out of unknown confined
362 water bodies within the experimental time. We interpret this difference as evidence for a divergent
363 cost and benefit trade off as jumping out of an isolated pool of a known environment carries less risk
364 than jumping in a novel environment.

365 We also found differences between female, male, and juvenile jumping behaviour. Juveniles jumped
366 less than adult fish, and females jumped faster than juveniles and with a stronger directional choice.
367 While we had no clear a priori prediction about how groups may differ in jumping behaviour, we
368 speculate how this difference may be explained. Divergence in risks of jumping may be one
369 explanation; for juveniles, the risk of jumping may be greater, since a smaller body is related to higher
desiccation and injury risk in fish (Odeh, 2002). Moreover, juveniles might lack landscape knowledge
370 and jumping skills, which increases the chance of hitting a rock. Differences in experience may also
371 explain the difference in latency time of jumping between males and males. Females in the wild are
372 less predated on and live longer than males (Reznick and Bryant 2007; Reznick et al. 1996). Due to
373 their increased lifespan, they can gather more landscape knowledge and jumping skills. In contrast,
374 males, having a shorter lifespan, might have evolved to invest more in mating efforts than in jumping
375
376
377
378
and orientation skills. This reasoning is supported by our findings that while all fish showed the same orientational pattern when exposed to matching cues, only females significantly favoured one direction over another. However, this line of argument seems to oppose a previous study that found that in mazes male guppies have better spatial abilities (Lucon-Xiccato & Bisazza, 2017c). The authors suggest that because males have larger home ranges and disperse further (Croft et al., 2003; Croft, Krause, & James, 2004; Darden & Croft, 2008), they have evolved better spatial cognition. Additional data is clearly needed to clarify how maze performance relates to orientational skills in the wild, focusing in particular on jumping behaviour.

To conclude, this study contributes to our understanding of how fish are able to use different cues to find suitable water bodies. In a lab experiment, we found that guppies were more likely to jump towards deeper areas when they are in a familiar entrapment, hence incorporating past spatial information to jump to safety. In a matched vs. mismatched spatial cue experiment in the field, we found that animals only showed directed jumping when general visual and auditory cues matched. Hence, in this study, we show that fish rely on the use of cognitive maps and a combination of general landmarks to orient themselves during aerial jumping.

394
395 Acknowledgements

396 We thank Amy Deacon for assistance during the field trip in Trinidad, and Bart Pollux for providing
397 stock fish to start our experimental populations at WUR. We further thank the staff from the BHE
398 group and Carus at WUR for technical and husbandry assistance.

399 Funding information

400 This research was funded by grants from the Foundation Lucie Burgers for Comparative Behavior
401 Research, Arnhem, the Netherlands, the Royal Netherlands Academy of Arts and Sciences
402 (KNAWWF/DA/973/Eco2013) to CVP, a Carl Tryggers Stiftelse (CTS18:205) to AK, and RV-T was
403 funded by a Biotechnology and Biological Sciences Research Council (BBSRC) Grant
404 (BB/V001256/1).

405 Contributions

406 C.V.P., R.V.-T., D.J.M., D.v.B., E.L., L.M.L., and A.K. conceived the study. C.V.P., R.V.-T., D.J.M.,
407 D.v.B., E.L., and L.M.L. collected the data. H.D.W, D.v.B., and E.L. analysed the data and wrote the
408 first version of the manuscript. All authors provided feedback on earlier versions of the manuscript
409 and contributed to its final version.

410 Ethical considerations

411 The care and use of experimental animals complied with animal welfare laws, guidelines and policies
412 as approved by the Animal Welfare Body (Instantie voor Dierenwelwijn, IvD) at Wageningen
413 University and Research and by the Fisheries Division of the Ministry of Agriculture, Land and
414 Fisheries of Trinidad and Tobago. Experiments involved behavioural observations, and stress to the
415 animals was minimised as much as possible. All wild fish used in the experiments were released
416 unharmed into the stream, and experimental females in the lab were returned to stock populations.

417
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**Figure legends**
**Figure 1.** Set up of the experimental tank: view from above (A), and view from the side (B). The experimental tank was filled with gravel to create four quadrants of different depths (two shallow areas, two deep areas). Two- cm ridges separated the quadrants from each other. Different shades of blue represent different depths, with darker tones marking the deeper areas. The “T” marks the testing arena. 7

**Figure 2.** Schematic overview of the study site. Test locations are marked A through E. At each location both treatments (M: matching cues; C: contrasting cues) were tested. 9

**Figure 3.** Percentage of jumps (A) and latency to jump (B) for guppies jumping into the deep areas (dark blue) and onto the shallow areas (light blue). Black markers in B show individual data points. 12

**Figure 4.** Latency to jump for the different treatments (contrasting cues and matching cues) (A) and for females, males, and juveniles (B). The proportion of jumpers for each group is noted underneath the violin plot. Horizontal lines indicate medians, boxes indicate interquartile range, and whiskers indicate all points within 1.5 times the interquartile range. 13

**Figure 5.** Jumping distribution for (A) contrasting cues (n = 139) and (B) matching cues (n = 136). The arrow points towards the mean angle and its length reflects the concentration of data. Black dots indicate individual data points. 14

**Figure 6.** Jumping distribution for the different stages (female, male, and juvenile) for contrasting cues (A) and matching cues (B). The arrow points towards the mean angle and its length reflects the concentration of data. 15