Experimental fish acquisition
In March 2017 and 2019, a hybridization experiment and parental reproduction were performed; details on the methods can be found in the literature [27]. Age-two fishes (PP (122.03 ± 1.78 mm, 25.2 ± 1.05 g), SW (106.78 ± 1.41 mm, 18.43 ± 0.74 g) and PS (125.84 ± 2.71 mm, 29.22 ± 1.85 g)) were used to quantify both external and skeletal characteristics, and age-one fishes (PP (9.08 ± 0.34 mm, 12.07 ± 0.90 g), SW (9.23 ± 0.14 mm, 13.03 ± 1.50 g) and PS (9.17 ± 0.48 mm, 14.02 ± 3.76 g)) were used to quantify foraging and behavioural features.
Food types
We used two types of food corresponding to parent resources. The first type consisted of small fishes, Sinibrama taeniatus (0.0507 ± 0.0043 g) or Carassius auratus (0.0748 ± 0.0023 g), the former of which is mainly distributed in the upper Yangtze River and the latter of which is widely distributed. The second type consisted of periphytic algae (Spirogyra, tough population on the pool wall or tender population on stone. Unfortunately, we did not produce the most palatable diatoms for SW in the pond; however, Schizothorax fishes, such as SW, still eat a certain amount of Spirogyra algae under natural conditions [34]), it is widely distributed and abundant in China’s water system.
Morphology
The external morphology of age-two SW (n = 30), PP (n = 30) and PS (n = 30) was studied, and the examination standards are shown in Additional Table 2. Then, we random selected 10 fish individuals from each species for quantification of skeletal morphology. Their opercular bone, pharyngeal bone, dentary bone and skull were obtained by boiling, and the examination standards are described in Additional Fig. 1. Finally, 19 external morphological indicators and 19 skeletal morphological indicators were quantified in this study, as shown in Additional Tables 2–3. To visually show the comprehensive morphological differences between the three fishes, we conducted principal component analysis (PCA) of two categories of indicators (Additional Tables 4–7).
The body shapes were photographed using an SLR camera (Canon EOS 100D, Japan). The details of the heads fixed by Bouin’s fixative and bones were photographed (Fig. 1) by a stereomicroscope (Nikon SMZ25). Age-two PP, SW and PS were scanned (Fig. 2) using a MicroCT Skyscan 1176 (Bruker, Belgium) to obtain the holistic bone structure; specific methods are described in [35], and they were slightly modified in this study.
Comparison of foraging habit
We fed PP, SW and PS with small fishes (S. taeniatus) and tough periphytic algae on the pool wall (Fig. 3). After the experimental fish had adapted to the food for a period of time, we dissected them and weighed their chyme. Specific experimental methods can be found in Additional method 1. We compared each fish species’ foraging level (FL) using the following formula:
$$ FL=M2/\left(M1-M2\right) $$
where M1 represents body weight, and M2 represents chyme weight.
Due to the large number of quantitative indicators in this study, such as FL, the contents and abbreviations of all the quantitative indicators are shown in Additional Table 1 for the convenience of readers.
Hybrid vs P. pingi in foraging fish
We compared the foraging capacity of PP (n = 15) and PS (n = 18) for small fishes (S. taeniatus) (Fig. 4a). Specific experimental methods are described in Additional method 2. We observed experimental fishes by video and quickly replayed the video and counted the following indicators: first attack time (FAT), first success time (FST), the success rate of the first successful capture (SRFC), first attack time after the first successful capture (FAT2), attack frequency (AF), the success rate of the total attacks (SRTA), and the spitting rate (SR). Details of these indicators are as follows:
FAT: The time when an experimental fish first attacked the small fishes. To exclude the influence of irritability, only the experimental fishes that launched the first attack within 5 min were included in all statistical comparisons.
FST: The time when an experimental fish first successfully caught a small fish. If it did not succeed within 30 min, a value of 30 min was used as its first success time.
SRFC: The success rate when an experimental fish first successful capture.
FAT2: The time when an experimental fish first attacked after the first successful capture.
AF: The average number of attacks per minute of an experimental fish; this value was calculated using the following formula:
SRTA: This value was calculated using the following formula:
SR: Some individuals catch fish and then spit them out; this value was calculated using the following formula:
$$ SR=N^{\prime\prime }/N^{\prime } $$
where N represents the total number of attacks; T represents the time at the end of the experiment; N′ represents the total catch before the end of the experiment (not intake); and N ′ ′ represents the number of fish spitted.
We compared the abilities of SW (n = 16) and PS (n = 20) to forage tender periphytic algae (Fig. 4b). Specific experimental methods are described in Additional method 3. We quickly replayed the video and evaluated the following indicators: FAT, AF, FL and foraging efficiency (FE). The details of these indicators are as follows
FAT: The time when an experimental fish first scraped periphytic algae from the rocks.
AF: The average number of scrapings per hour of experimental fish; this value was calculated using the following formula:
$$ AF=\left(N2+N5+N8\right)/3 $$
FE: The average weight of a single scrape of periphytic algae per unit weight of experimental fish; this value was calculated using the following formula:
$$ EF=M2/\left( AF\times 8\times \left(M1-M2\right)\right) $$
where N2, N5, and N8 represent the number of attacks in the second, fifth and eighth hours, respectively, M1 represents the body weight of the experimental fish; and M2 represents the chyme weight of the experimental fish.
Assessment of whether the behaviour of hybrid fish spitting fish is persistent
In the previous experiments, we observed that PS had obvious behaviour of spitting fish (Fig. 3c and Additional Movie 3). To test if this behavior is persistent, we set up a feeding experiment using small fish (C. auratus (Fig. 5a)) for 9 days, and PS still had obvious spiting behaviour after catching the small C. auratus fishes (Fig. 5c). For 9 days, we fed not only fish but also blood worms (Fig. 5b, 0.0171 ± 0.0006 g, Chironomidae larvae, a soft-bodied aquatic insect) to simulate a palatable food shortage, but not a complete absence, in the natural environment. Specific experimental methods are described in Additional method 4. We counted the daily catch, intake, and spitting of each PS for small fish.
Additional file 3: Movie 3.
Mechanism explaining why hybrid fish spitted fish
Two mechanisms may explain why PS spitted small fish: the small fish tasted bad or they were difficult to chew. To explore this mechanism, we selected approximately 50 g of C. carp (Fig. 6a) and cut the back muscle into small pieces (Fig. 6b) without bone, instead of using small fish. We took PS that had the obvious behaviour of spitting small fish in the last experiment as the experimental fishes (n = 7). Other than the small fishes that were replaced with small pieces of C. carp muscle, the other feeding and statistical schemes were the same as those in Section 2.7. However, the experiment lasted only 3 days. We counted the average number of daily foraging (ANDF) and the SR of the 7 experimental fishes used in Section 2.7 and this experiment, which was equivalent to the former serving as a control group for the latter, by the following formulas:
where N represents the total number of prey captured by PS during the experiment, and T represents the number of days of the experiment.
Next, to investigate whether prey size also leads to fish-spitting behaviour in PS, the SR of PS to different sizes of meat and fish was quantified. The specific experimental methods are described in Additional method 5.
Then, we compared the pharyngeal teeth details of PP, SW and PS and quantified the maximum opening distance between their pharyngeal teeth and their puncture ability based on the following principle: for the same pressure and a smaller force area, the greater the pressure. We quantified the following indicators: the maximum opening width between pharyngeal teeth (MOWPT), the development degree of hook pharyngeal teeth (DDHPT) and the grinding surface area of pharyngeal teeth (GSAPH); these values were calculated using the following formulas:
$$ DDHPT= TL^{\prime }/ TL $$
$$ GSAPH=S^{\prime }/S $$
where TW represents the maximum width distance between pharyngeal teeth, HW represents head width; T represents average length of 5 lateral pharyngeal teeth, TL′ represents the average length of the hooked portion at the tip of the lateral 5 pharyngeal teeth; and S represents the basal area of all pharyngeal teeth, S′ represents the grinding surface area of all pharyngeal teeth. Further information on these parameters is provided in Additional Figure 2.
We quantified the foraging-related traits (Additional Table 8, 20 measured traits and 17 standardized traits) of all fishes (n = 32) in Section 2.7 to explore whether a correlation exists between these traits, and these indicators included the TNC (the total number of captures), TNI (the total number of ingestions), TNSF (the total number of spitting fish) and SR by Spearman’s correlation in SPSS 21.0.
Statistical analyses
The mean ± standard deviation (SD) was used to represent the unannotated quantitative data, and the other data are annotated in the table or graph notes. One-way analysis of variance (ANOVA) was used to analyse the data of three independent experiments. Spearman’s correlation method was used to analyse the correlation. All the data obtained above were measured and calculated using SPSS software version 19. Tukey’s test was used to analyse the difference. We conducted principal component analysis (PCA) of the Z-scores of these indicators using IBM SPSS Statistics (version 21.0, Armonk, New York, United States). All graphs were generated by the Origin software version 2019b or SPSS software version 19.