Stomach contents of Great Cormorants
A total of 13 fish species, including 102 individuals and two prawn (Macrobrachium nipponense and digested Caridean species), were extracted from 19 cormorants culled in the colony (Table 2). The detailed information about the cormorants having contained the prey in the stomach (Table S1) and the respective prey species (Table S2) is shown in Electronic Supplementary Material (ESM). Ayu (Plecoglossus altivelis altivelis), the most important mass-release fish in the watershed, was found in the stomachs of seven cormorants with relatively high occurrence frequency (9.6%). The occurrence of Japanese dace (T. hakonensis) in the cormorant stomachs was also relatively frequent with a frequency of 8.2% (a total of six cormorants). We also found two mass-release species, amago trout (Oncorhynchus masou ishikawae) in three cormorants and rainbow trout (Oncorhynchus mykiss) in two cormorants.
The dace (T. hakonensis) was conspicuous in total number of individuals extracted from the cormorant stomachs (n = 19) and the total wet weight (348.3 g) (Table 2). The rainbow trout (O. mykiss) was also abundant in the total weight (289.6 g), but not in total number of the extracted individuals (n = 3). By contrast, the ayu (P. altivelis altivelis) totaled 95.4 g only in weight, but was relatively high in the occurrence frequency (9.6%) and total number of the extracted individuals (n = 15). The amago trout (O. masou ishikawae) (145.5 g) was also not much in weight in disproportion to the catch of relatively many individuals (n = 12).
The body length (standard length) of the stomach-content fish ranged from 32.6 mm in Japanese fluvial sculpin (Cottus reinii) to 350.0 mm in rainbow trout (O. mykiss). There were of course damaged fish that could not be measured for standard length. The sizes of the ayu (P. altivelis altivelis) and the dace (T. hakonensis) extracted from the stomachs were similar, with 92.8–139.3 mm in the ayu and 86.2–171.2 mm in the dace.
Distributions of fish in the Kano River watershed
A total of 30 fish species including 4980 individuals were collected by means of throw nets in the Kano River watershed from July 2008 to December 2009 (Table 3, Appendices 1–5 in the ESM). Two cyprinid juveniles could not be identified. The fish consisted mainly of Cyprinids (n = 3803) and Salmonids (n = 496), occupying 76.4% and 10.0% of all the 4980 individuals, respectively. The result in our research is largely consistent with the result of the fish fauna research in the 1970 s by Itai (1982).
Downstream fatminnow (Rhynchocypris lagowskii) were most frequently collected in the throw-net sampling, mostly distributed in the tributaries of the entire watershed (20 stations). In combination with the result in the supplementary sampling, the downstream fatminnow were collected at 48 stations (Fig. 2a). The total catch of this species in the throw-net sampling was the third most abundant with 717 individuals.
The dace (T. hakonensis) was also collected at many stations (17 stations) in the throw-net sampling, with the most abundant catch of 1810 individuals (Table 3). The dace was ubiquitously caught in both the main stream and the tributaries, with the catch at 23 stations in the total sampling (Fig. 2b).
The amago trout (O. masou ishikawae) (12 stations), pale chub (Zacco platypus) (11 stations), and the ayu (P. altivelis altivelis) (10 stations) were also collected with throw nets at relatively many stations. The amago trout were mostly collected in the upper reaches of the mainstream and the tributaries within the Izu Peninsula (Fig. 2c). The total catch of this species in the throw-net sampling was also abundant, specifically fourth in abundance (n = 428, Table 3). The pale chub and the ayu were mostly collected in the mainstream and the northeastern tributaries (Fig. 2d, e), becoming the second most (812 individuals) and the fifth most (217 individuals) abundant catch in the throw-net sampling (Table 3), respectively.
Amur goby (Rhinogobius nagoyae) and Japanese fluvial sculpin (C. reinii), extracted from the stomachs of 2–3 cormorants (Table 2), were mostly collected in the mainstream (Fig. 2f, g). The rainbow trout (O. mykiss), mass-release species, were collected in the northern and northeastern tributaries only (Fig. 2h).
Stable isotope ratios of Great Cormorants
Stable isotope ratios of the cormorant tissues ranged from − 23.0 to − 14.7‰ in δ13C and 8.9–18.4‰ in δ15N for defatted muscles and − 23.9 to − 13.9‰ in δ13C and 9.8–19.4‰ in δ15N for defatted livers (Table 4, Fig. 3a). The isotopic values of the livers were significantly higher than the values of the muscles for both δ13C and δ15N (Wilcoxon signed rank test; δ13C, P < 0.0001; δ15N, P < 0.0001).
There are not clear isotopic differences in the cormorant tissues among years (2009–2011) in either muscles or livers as shown in Fig. S1 (ESM). We performed Kruskal–Wallis tests and the subsequent Dunn’s multiple comparison tests for four categories, δ13C and δ15N of muscles and livers, respectively. In the result, there was no significant difference among the 3 years in every category (Kruskal–Wallis tests, P > 0.05), and there was no significant difference in every combination of the years in every category (Dunn’s multiple comparison tests, P > 0.05).
Furthermore, clear differences were neither found between different developmental stages nor between sexes (Figs. S2, S3 in the ESM). We performed Kruskal–Wallis tests and the subsequent Dunn’s multiple comparison tests for four categories, δ13C and δ15N of muscles and livers, respectively. In the result, there was no significant difference among four groups, the adults and the young of females and males, in every category (Kruskal–Wallis tests, P > 0.05), and there was no significant difference in every combination of the groups in every category (Dunn’s multiple comparison tests, P > 0.05).
Mizutani et al. (1991) experimentally demonstrated that the diet of Great Cormorants is 2.1‰ lower in δ13C and 2.4‰ lower in δ15N than the muscle of the cormorant and 1.3‰ lower in δ13C and 2.3‰ lower in δ15N than the liver of the cormorants. Subtracting these values from the values of the cormorant tissues, the assumed isotopic values of the past diets were calculated to be − 25.1 to − 16.8‰ in δ13C and 6.5–16.0‰ in δ15N for muscle-base calculation, and − 25.2 to − 15.2‰ in δ13C and 7.5–17.1‰ in δ15N for liver-base calculation (Fig. 3b). The liver-based dietary values were significantly higher than the muscle-based dietary values in both δ13C and δ15N (Wilcoxon signed rank test; δ13C, P < 0.0001; δ15N, P < 0.0001).
According to the statistical comparison between the defatted tissue and the untreated tissue of the identical cormorant (n = 30), both δ13C and δ15N were significantly higher in the defatted tissue than in the untreated tissue (Table 5, Wilcoxon signed rank test, P < 0.0001 in both δ13C and δ15N for the muscle and the liver respectively). The differences in the averages of the δ13C were 1.1 ± 0.4‰ for the muscle and 1.2 ± 0.5‰ for the liver, slightly larger than the differences of the δ15N with 0.3 ± 0.2‰ for the muscle and 0.2 ± 0.2‰ for the liver.
Stable isotope ratios of ayu
The stable isotope ratios of the ayu (P. altivelis altivelis) collected with throw nets ranged − 28.5 to − 12.2‰ in δ13C and 4.4–12.6‰ in δ15N (69.4–152.8 mm body length, Table 6, Fig. 4a). The isotopic distribution of the ayu was markedly different between the northeastern tributary (H1 and H2) and the mainstream (C1–C6) (Fig. 5). The ayu from the northeastern tributary were characterized by low δ13C values of − 28.5 to − 19.8‰, whereas the ayu from the mainstream was characterized by high δ13C values of − 19.8 to − 12.2‰ and low δ15N values of 4.4–10.8‰, except for five individuals from C1 and an individual from C3. The ayu collected at E2, a tributary station neighboring on the mainstream, overlapped in δ13C and δ15N with the ayu from the mainstream.
The stable isotope ratios of the ayu extracted from the stomachs of the culled cormorants ranged from − 18.6 to − 10.9‰ in δ13C and 6.8–14.3‰ in δ15N (Table 7). In the δ13C–δ15N map, the distribution of the stomach-content ayu partly overlapped with the distribution of the ayu collected with throw nets in the mainstream (C2–C6) (Fig. 5).
The stable isotope ratios of the mass-release ayu ranged from − 18.1 to − 17.3‰ in δ13C and 11.9–13.2‰ in δ15N for the individuals released on 15 March 2013 and ranged from − 17.4 to − 17.0‰ in δ13C and 13.8–14.2‰ in δ15N for the individuals released on 16 April 2013 (Table 8, Fig. 5). The δ13C averaged − 17.7 ± 0.2‰ in March and − 17.2 ± 0.1‰ in April, 1.2–1.7‰ higher than the δ13C value of − 18.9‰ for formula feed supplied to the rearing pond. There was a significant difference between March and April (Mann–Whitney’s U-test, P < 0.0001). The averages of δ15N in March (12.5 ± 0.5‰) and April (14.0 ± 0.2‰) were also higher than the δ15N value of 12.1‰ for the formula feed. In the δ15N, the difference between the formula feed and the mass-release ayu (averages) was larger in April (1.9‰) than the difference in March (0.4‰). According to Mann–Whitney’s U-test, a significant difference was found for the δ15N of the ayu between March and April (P < 0.0001).
Stable isotope ratios of Japanese dace
The stable isotope ratios of the dace (T. hakonensis), which was a conspicuous species in the stomach contents of the culled cormorants, were analyzed for the individuals collected with throw nets in the Kano River watershed. The isotopic values of this species ranged from − 25.4 to − 14.3‰ in δ13C and 4.3–13.6‰ in δ15N (40.5–158.9 mm body length, Table 9). There were marked differences among the sampling stations.
The isotopic variety in the dace from the watershed was mainly characterized by local differences among tributary stations, with low δ13C values (− 25.4 to − 23.8‰) for Stn. E1, high δ13C values (− 19.6 to − 14.3‰) for N1, low δ15N values (4.3–5.5‰) for W3, and high δ15N values (12.0–13.6‰) for N2–N5 (Fig. 4b). On the other hand, the stable isotope ratios of the dace extracted from the stomachs of the culled cormorants ranged from − 22.6 to − 14.2‰ in δ13C and 6.6–13.1‰ in δ15N (Table 7). In the δ13C–δ15N map, the distribution of the stomach-content dace overlapped with the distribution of the dace collected by the throw-net sampling (Fig. 6). Most of the stomach-content dace overlapped with the dace from the mainstream (C1–C6) and its neighboring tributary station (E2), the northern tributary (N1–N5), and the northeastern tributary (H1–H3).
Only two dace extracted from a cormorant showed a peculiar isotopic distribution (− 16.1 to − 15.2‰ in δ13C and 12.5–13.1‰ in δ15N), separating from the dace collected in the Kano River watershed (Fig. 6). This isotopic distribution is close to the isotopic values of Japanese horse mackerel (T. japonicus) collected in the river mouth of the Kano River, and the dace collected in the river mouth of the Inohzawa River (Table 9).