Effects of salinity on otolith Sr:Ca ratios
The mean total lengths (TLs) in the four experimental salinities of 0, 10, 20 and 30 psu were 17.3 ± 0.5 (± SD) mm, 18.2 ± 1.3 mm, 18.6 ± 0.7 mm and 17.5 ± 0.8 mm, respectively. There were significant differences in TL among the four salinities (Kruskal–Wallis test, n = 40, H = 13.577, p < 0.005), and were found between 0 and 10 psu and 20 psu and between 20 and 30 psu (Mann Whitney-U test, df = 11–17, U = 6–20, p < 0.05–0.001). No significant differences were found for the other combinations (Mann Whitney-U test, df = 14–15, U = 33–42, p > 0.05).
The mean Sr and Ca concentrations in the rearing waters ranged from 0.03 ± 0.001 (± SD) mg l-1 (0 psu) to 8.12 ± 0.3 mg l-1 (30 psu) and from 4.97 ± 0.15 mg l-1 (0 psu) to 336 ± 28.3 mg l-1 (30 psu), respectively. The relationships between salinity (S) and Sr and Ca were Sr = 0.269S–1.17 (r2 = 0.996) and Ca = 10.99S–54.96 (r2 = 0.998), respectively. The Sr and Ca concentrations in the rearing water differed significantly among the four experimental salinities (Kruskal–Wallis test, n = 80, H = 74.967, p < 0.0001), and they both significantly increased with salinity (t-test, df = 80, p < 0.0001). The mean Sr:Ca ratios in the rearing waters ranged from 2.44 × 10–3 ± 0.08 × 10–3 (0 psu) to 9.64 × 10–3 ± 0.82 × 10–3 (30 psu). The relationship between salinity (S) and the Sr:Ca ratio in the water was water Sr:Ca ratio = 1.27S + 9.52 (r2 = 0.852) and the Sr:Ca ratio in the rearing water significantly increased with salinity (t-test, df = 80, p < 0.0001).
There were significant differences of the Sr:Ca ratio in the otoliths among the four experimental salinities (Kruskal–Wallis test, H = 22.813, p < 0.0001). The mean (± SD) Sr:Ca ratio in the otoliths significantly increased from 1.20 × 10–3 ± 0.98 × 10–3 in 0 psu to 4.30 × 10–3 ± 0.38 × 10–3 in 30 psu, and the Sr:Ca ratio in the otoliths was significantly correlated with the salinity (otolith Sr:Ca ratio = 0.09S + 2.13, r2 = 0.708; t-test, df = 39, p < 0.0001). These results suggest that the otolith Sr:Ca ratio in threespine sticklebacks is affected by ambient salinity.
The mean otolith Sr:Ca ratio in the wild sticklebacks that lived in an artificial freshwater pond was 1.31 × 10–3 ± 0.10 × 10–3 (± SD). No significant difference was found in the otolith Sr:Ca ratios between reared and wild sticklebacks in the freshwater environment (Mann Whitney-U test, df = 9, U = 47, p > 0.05). According to the linear regression between otolith Sr:Ca ratio and salinity and the otolith Sr:Ca values of freshwater in the reared and the wild sticklebacks and phase L (low Sr:Ca ratio) in the wild sticklebacks examined migratory history (see Migratory history), < 2.5 × 10–3 was used as an indicator of a freshwater environment (freshwater resident), distinct from a brackish or marine environment, in the present study. We also categorized the specimens into “anadromous” which had a transition point (TP) from phase-L (low Sr:Ca ratios < 2.5 × 10–3) to phase-H (high Sr:Ca ratios ≥ 2.5 × 10–3) along the line history transect and “estuarine resident” which showed constantly high Sr:Ca ratios (≥ 2.5 × 10–3) from the otolith core to the edge, followed in previous26,27 and present studies.
Migratory history
There were three migratory types, i.e., freshwater resident, anadromous and estuarine resident, in Gasterosteus aculeatus (Fig. 2). The freshwater residents showed consistently low Sr:Ca ratios along a line transect, averaging 1.39 × 10–3 ± 0.22 × 10–3 (± SD) (range: 1.14–1.80 × 10–3) (Fig. 2a), characterized by a bluish colour (low Sr concentration) from the otolith core to the edge in the otolith X-ray intensity map (Fig. 3a). We found 35 of 144 (24.3%) specimens were freshwater residents, suggesting continuous residence in freshwater environments after hatching, although the habitats (Otsuchi and Kozuchi rivers) directly flow to the sea (Otsuchi Bay) and the fish can migrate downstream to the sea (Fig. 1). The anadromous showed a low Sr:Ca ratio phase from the core to the point approximately 170–200 µm (phase L), averaging 1.93 × 10–3 ± 0.41 × 10–3 (± SD) (range 1.26–2.38 × 10–3), which corresponds to freshwater life period. Thereafter, the ratios increased sharply, averaging 4.96 × 10–3 ± 0.51 × 10–3 (± SD) (range 4.13–6.15 × 10–3) and were maintained at higher levels until the outermost regions corresponded to a brackish water or seawater life period (phase H, Fig. 2b). Significant differences occurred in the Sr:Ca ratios between phase L and phase H in the 17 anadromous specimens (Mann Whitney-U test, df = 84–180, U = 41–1,236, p < 0.0001). The anadromous showed a wide space of bluish colour (low Sr) radiating from the centre, which was surrounded by concentric rings having higher Sr concentrations in the otolith X-ray intensity map (Fig. 3b), which corresponded to the otolith Sr:Ca ratios along a line transect. We found 11.8% (17 of 144) sticklebacks showed a typical anadromous migration history, having a clear transition point (TP) from phase L to phase H in a line transect (Fig. 2b). The estuarine residents, which did not have a clear TP along a life history transect, showed relatively high Sr:Ca ratios consistently from the core to the edge, averaging 4.94 × 10–3 ± 0.53 × 10–3 (range 4.55–5.93 × 10–3) (Fig. 2c). The estuarine residents were characterized by reddish and greenish colours (higher Sr concentration) from the otolith core to the edge in the otolith X-ray intensity map (Fig. 3c). We found 92 of 144 (63.9%) sticklebacks showed consistently high Sr:Ca ratios along a line transect, although these specimens were classified either as anadromous or freshwater residents based on morphology. Freshwater resident and anadromous sticklebacks as inferred by morphology and the habitat environments occurred sympatrically in Hyotan Marsh, and the TL in the freshwater residents (mean ± SD; 70.1 ± 3.7) was significantly smaller than that of the anadromous (87.5 ± 4.7) (Mann Whitney-U test, df = 13, U = 11, p < 0.0001), while their life histories were both estuarine residents (Fig. 2c, Fig. 3d,e, Table 1). Interestingly, otolith Sr:Ca ratios from the Hyotan Marsh of the morphologically freshwater resident type showed a slight increase around between 150 and 250 μm from the core, and then it decreased again to the otolith edge (Fig. 2c, Fig. 3d). The mean TLs of freshwater residents, anadromous and estuarine residents in all G. aculeatus specimens as determined by otolith Sr:Ca ratios were 68.6 ± 5.8 mm (mean ± SD), 88.9 ± 6.2 mm and 87.6 ± 8.7 mm, respectively. There were significant differences in mean TLs among the three migration types (Kruskal–Wallis test, n = 143, H = 65.568, p < 0.0001). Significant differences were found among freshwater residents and anadromous and estuarine residents (Mann Whitney-U test, df = 27–91, U = 2–164, p < 0.0001) while no significant difference was found between anadromous and estuarine resident (Mann Whitney-U test, df = 26, U = 727.5, p > 0.05).
The locations where the threespine sticklebacks, Gasterosteus aculeatus and G. nipponicus, were collected. Map of sampling locations of the threespine sticklebacks, G. aculeatus and G. nipponicus in northern Japan. Numbers with the black dots on the map of northern Japan (upper left corner) correspond to each sampling site. 1, Cape Soya, 2, Toyokanbetsu River, 3, Lake Saroma, 4, Biwase River, 5, Biwase River tidal pool, 6, Hyotan Marsh, 7, Shiomi River, 8, Obetsu River, 9, Akkeshi Bay, 10, Numajiri River, 11, Lake Takkobu, 12, Lake Ogawara, 13, Miyako Bay, 14, Funakoshi Bay, 15, Otsuchi Bay, 16, Otsuchi River, 17, Kozuchi River, 18, Okirai Bay. The base map was downloaded from the USGS National Map Viewer (open access) at https://viewer.nationalmap.gov/viewer/ and from the OpenStreetMap (open access) at https://www.openstreetmap.org.
Migratory history of threespine stickleback G. aculeatus as indicated by the temporal pattern of the Sr:Ca ratios in their otoliths. Plots of otolith Sr:Ca ratios along a transect line from the core to the edge of the otolith were divided into freshwater resident (a), anadromous (b) and estuarine resident (c). Life history type, as determined by morphological characteristic, is indicated in the upper right corner in each plot.
Two-dimensional images made using X-ray electron microprobe analysis of the Sr concentrations in the otoliths of threespine sticklebacks Gasterosteus aculeatus and G. nipponicus in northern Japan. Freshwater resident (a) migration pattern of a G. aculeatus morphologically freshwater resident specimen, anadromous (b) and estuarine resident (c) migration patterns in G. aculeatus of morphologically anadromous specimens, estuarine resident migration patterns in G. aculeatus of morphologically freshwater resident (d) and anadromous (e) specimens collected in Hyotan Marsh and anadromous (f) and estuarine resident (g) migration patterns in G. nipponicus of morphologically anadromous specimens. Sr concentrations are represented by 16 colours from red (highest) to yellow to green to blue (lowest).
In G. nipponicus, there were two migratory types, i.e. anadromous and estuarine residents, as determined from otolith Sr:Ca ratios along a line transect and otolith X-ray intensity maps of the otolith Sr content (Figs. 3, 4). Otolith micochemical signatures could identify anadromous among a total of 56 in 477 specimens, and all of the rest of sticklebacks (421 specimens) were estuarine residents (Table 1). We found 56 of 477 (11.7%) sticklebacks had a TP at approximately 150–190 µm along a line transect and showed typical anadromous (Fig. 4a). The mean Sr:Ca ratios in phase L and phase H averaged 1.88 × 10–3 ± 0.32 × 10–3 (± SD) (range: 0.97–2.23 × 10–3) and 5.24 × 10–3 ± 0.42 × 10–3 (range: 4.21–6.43 × 10–3), respectively, and those phases were significantly different (Mann Whitney-U test, df = 18–185, U = 0–715, p < 0.0001). Two-dimensional images of the Sr concentration in the otoliths showed a wide space of bluish colour (low Sr) radiating from the centre, which was surrounded by higher Sr concentrations (Fig. 3f). In Lake Takkobu, a freshwater lake, 28 of 30 sticklebacks showed the anadromous, and a slight decrease in otolith Sr:Ca ratio was found around the edge in 16 of the 28 sticklebacks. Ten of the 16 fishes had an otolith Sr:Ca ratio less than 2.5 × 10–3 (mean ± SD: 1.85 × 10–3 ± 0.56 × 10–3, range: 0.75–2.41 × 10–3). The low Sr:Ca ratio in the otolith edge corresponds to a short freshwater period during spawning migration in G. nipponicus. The estuarine resident was the dominant migratory pattern, constituting 88.3% (421 of 477) of the sticklebacks. The estuarine residents showed consistently high Sr:Ca ratios from the core to the edge (Fig. 4b), averaging 5.58 × 10–3 ± 0.93 × 10–3 (range: 4.60–8.09 × 10–3), characterized by a higher Sr concentration throughout the whole otolith in the otolith X-ray intensity map (Fig. 3g). Although G. nipponicus is thought to be only an anadromous based on its morphology and habitat environments, most of the sticklebacks showed an estuarine resident without any signs of freshwater life (Fig. 4b), similar to G. aculeatus.
Migratory history of threespine stickleback Gasterosteus nipponicus as indicated by the temporal pattern of the Sr:Ca ratios in their otoliths. Plots of otolith Sr:Ca ratios along a transect line from the core to the edge of the otolith were divided into anadromous (a) and estuarine resident (b).
There were no significant differences in the mean Sr:Ca ratios of estuarine residents between G. aculeatus and G. nipponicus (Mann Whitney-U test, df = 15, U = 26, p > 0.05). No significant differences in the mean Sr:Ca ratios of phase L and phase H in anadromous were also found between species (Mann Whitney-U test, df = 21–23, U = 279–457, p > 0.05). These results suggested there is no significant interspecific variation in otolith Sr:Ca ratio.
Habitat use
The mean otolith Sr:Ca ratios from the core to the edge in freshwater resident, anadromous and estuarine resident were 1.39 × 10–3 ± 0.22 × 10–3 (mean ± SD), 3.39 × 10–3 ± 0.45 × 10–3 and 5.13 × 10–3 ± 0.68 × 10–3, respectively. There were significant differences in the mean otolith Sr:Ca ratios among the three migration types (Kruskal–Wallis test, n = 622, H = 261.013, p < 0.005) and significant differences were found in all combinations (Mann Whitney-U test, df = 90–127, U = 0–556, p < 0.0001). The mean otolith Sr:Ca ratios can be used as a new indicator to differentiate habitat use in threespine sticklebacks because morphological analyses do not necessarily reflect their life history patterns (Table 1).
Gasterosteus aculeatus in the Otsuchi and Kozuchi rivers in complete freshwater environments above the intertidal areas were all freshwater residents (100%) (Fig. 5). Morphologically anadromous specimens of G. aculeatus were mostly estuarine residents, averaging 89.3 ± 11.8% (± SD) and ranging from 73 to 100% at each site (Fig. 5), while the remaining fishes (mean ± SD; 10.7 ± 11.8%, range 0% to 27%) were typical anadromous (Fig. 5). Interestingly, in Hyotan Marsh, otolith Sr:Ca ratios in morphologically freshwater resident fishes (mean ± SD; 6.0 × 10–3 ± 2.4 × 10–3) were significantly higher than those of morphologically anadromous fishes (5.1 × 10–3 ± 1.5 × 10–3) (Mann Whitney-U test, df = 11, U = 4, p < 0.01), although both sticklebacks were identified as estuarine residents by means of the otolith microchemical signatures.
Habitat use of threespine sticklebacks, Gasterosteus aculeatus and G. nipponicus, in northern Japan. Composition of habitat uses, i.e., freshwater resident, anadromous and estuarine resident, of G. aculeatus (right) and G. nipponicus (left) in each location. Blue: freshwater resident, yellow: anadromous, red: estuarine resident. Numbers with the black dot on the map of northern Japan correspond to each sampling site. 1, Cape Soya, 2, Toyokanbetsu River, 3, Lake Saroma, 4, Biwase River, 5, Biwase River tidal pool, 6, Hyotan Marsh, 7, Shiomi River, 8, Obetsu River, 9, Akkeshi Bay, 10, Numajiri River, 11, Lake Takkobu, 12, Lake Ogawara, 13, Miyako Bay, 14, Funakoshi Bay, 15, Otsuchi Bay, 16, Otsuchi River, 17, Kozuchi River, 18, Okirai Bay. The base map was downloaded from the USGS National Map Viewer (open access) at https://viewer.nationalmap.gov/viewer/.
In G. nipponicus, habitat use in 14 of 15 sites in morphologically anadromous was mostly estuarine resident, averaging 95.6 ± 6.4% (± SD) and ranging from 78 to 100% (Fig. 5), while only a few remaining individuals (mean ± SD; 4.4 ± 6.4%, range 0% to 22%) were actually anadromous (Fig. 5). Meanwhile, 93% of sticklebacks from the Lake Takkobu were typical anadromous, while 7% were estuarine residents.
The interannual variability of the habitat use was examined in Ohbetsu River (2 years) for G. aculeatus and Numajiri River (2 years) and Lake Ogawara (3 years) for G. nipponicus in the same period (Table 1, Fig. 5). The deviations of interannual variations were 5 to 12% in each site, and the composition of each habitat use was constant for several years. Furthermore, the occurrence of the “estuarine resident” novel life history found previously26,27 was temporally and spatially a general phenomenon in these threespine sticklebacks as their dominant migration pattern.
Source: Ecology - nature.com
