A total of 10 male reproductive processes (the process of preparing for spawning and subsequently performing egg care) were observed in 2 observation areas set up on the sandy bottom off Seisui and Katetsu (Fig. 2). Two males appeared in each observation area and no other male was observed in these areas. Although they were not tagged, at least the males at S1 and S3 and K1 and K3 were identified as the same individuals by the presence of lateral body scars in the former and the construction of another new circular structure during egg-care in the latter (Fig. 2). This is to say the actual number of male individuals in the study areas ranged from 4 (in the case that males at S1, S3 and S5; S2, S4 and S6; K1 and K3; and K2 and K4 were the same individuals, respectively) to 8 (in the case that all males except those at S1 and S3 and K1 and K3 were different individuals). Each of the males constructed a circular structure on the seabed (Fig. 3), which required 7–9 days to complete (Fig. 2; K1, K3 and K4). The behavioural patterns of the males changed during construction (Fig. 4).

Figure 2 Reproductive process of male pufferfish. S1–6 and K1–4 indicate circular structures found at Seisui and Katetsu, respectively. Columns of light green, green and blue indicate the duration of the early, middle and final stage of circular structure construction, respectively. Refer the text for the definitions of the 3 construction stages. Light blue columns indicate the duration of egg care. White stars indicate spawning, solid triangles indicate hatching. N, R and r indicate the number of peaks and valley pairs, radius and radius of the central zone of the circular structure, respectively. Full size image

Figure 3 Changes in the circular structure constructed by male pufferfish. (a) Early stage; (b) middle stage; (c) final stage; and (d) after spawning of the same circular structure of K1 in Figure 2. Photograph by Y. Okata on 23, 27, 29 June and 6 July 2012, respectively. Full size image

Figure 4 Changes in behavioural pattern frequency during construction of a circular structure. Columns in light green, green and blue indicate the duration of the early, middle and final stage of circular structure construction, respectively. See text for the definition of the 3 construction stages and the abbreviations of O-in, I-in, O-out, I-out, C and Dec. Data is based on the observations of K3 in Figure 2. O-in: P = 0.198 (N. S.), H = 3.236; I-in: P < 0.001, H = 15.676; O-out: P < 0.001, H = 15.973; I-out: P < 0.001, H = 25.805; C: P < 0.001, H = 28.956; Dec: P < 0.001, H = 21.253, Kruskal–Wallis test. Full size image

During the early stages of construction (on the day of and the day after construction of the new circular structure), each male created a basic circular shape (Fig. 3a). Males dug valleys in the sandy bottom using their pectoral fins, anal fins and caudal fins while swimming linearly (Fig. 1) and then swam at various angles in a radial direction from the outside of the circle to the inside, forming radially aligned peaks and valleys on the seabed. They passed from the outer zone towards the inside (O-in), the inner zone towards the inside (I-in) or continuously passed through both zones towards the inside (Figs. 4, 5, Supplementary Video 1). Males temporarily suspended fin flapping while moving inwards, thereby forming concavities and convexities in the valleys. They swam in random directions in the central zone (C), stirring the sand to form a flat area (Figs. 4, 5). In this way, a circular structure on the seabed composed of radially aligned peaks and valleys with a flat area at the centre was constructed. The radius of the circular structure and that of the final central zone measured 79–105 cm and 31–50 cm, respectively (n = 6, Fig. 2). The number of peak and valley pairs ranged from 24–32 (n = 10).

Figure 5 Sectional view of the radially aligned valley in the circular structure. As construction proceeded, the width of the central zone enlarged (dotted line). Full size image

During the middle stage of construction (between the early and final stages of construction), additional male behaviour refined the circular structure (Figs. 3b, 4). Valleys and peaks became more clearly defined as males repeatedly performed O-in and I-in behaviour (Supplementary Video 2). As the males swam in a circular direction and stirred the sand in the central zone (Supplementary Video 3), they flattened the seafloor surface and enlarged the width of the central zone (Fig. 5). Furthermore, when the males passed through the inner zone, moving outwards (I-out), fine sand particles were stirred up and flowed towards the central zone (Supplementary Videos 4, 5).

During the final stage of construction (on the day of and the day before spawning), the circular structure was completed. The most remarkable change that occurred during this stage was the formation of an irregular pattern in the central zone (Fig. 3c). This pattern was created by flapping the anal fin on the bottom of the central zone (Supplementary Video 6). The pattern was composed of the fine sand particles (Fig. 6a) that had been gathered in the central area. During this stage, I-out behaviour was not observed (Fig. 4); a part of the inner zone where males had been flapping their fins was flattened and the central zone was further enlarged (Fig. 5). Instead, they displayed flapping behaviour in the outer zone when they swam outward (O-out; Supplementary Video 6). During this behaviour, fine sand particles flowed towards the centre, similar to that during the I-out behaviour. During this stage, males also decorated the radially aligned peaks with shell and coral fragments and the frequency of the behaviour was significantly higher than that during other stages (Dec; Fig. 4). They chose fragments from the seafloor of the central zone and valleys and placed them on the peaks. Females were observed to visit the male's nest only during this stage of construction. When a female approached the circular structure, the male stirred up the sand particles in the central zone of the nest. When the female entered the nest, the male retreated from the central zone and rushed towards her several times (Supplementary Video 7). The female stayed in the nest for a minute and then left, often visiting the same nest again. Spawnings occurred in pairs and the eggs were released into the central zone of the nest.

Figure 6 Photomicrographs of the sand particles at the nest site. Collected on the day before spawning (a) and hatching (b). White bar = 5 mm. Full size image

After spawning, males remained in the circular structure for 6 days to care for the eggs (Fig. 2; S3, S4, K1 and K2). They did not perform digging or other maintenance behaviour of the radially aligned peaks or valleys during this period. As a result, the structure gradually collapsed and was smoothed to become almost flat by water currents (Fig. 3d). Furthermore, the fine sand particles were dispersed and replaced by coarser sand particles (Fig. 6b), which covered the nest site. After the eggs hatched, males left the nest site and soon reappeared in the observation area for the next reproductive cycle. However, they never returned to an old nest site but instead began to construct a new circular structure at a separate site.

A fluid dynamics experiment using a 1/2 scale model of the circular structure (Fig. 7a) revealed that strings placed upstream were turned towards the inner side (blue arrow) and those that were downstream turned towards the outer side of the structure (green arrow) and those on top of the hills and central zone were turned towards the downstream direction (deep blue arrows). The directions of strings in the valleys situated at the midpoint of upstream and downstream were unclear (light blue arrow). These results revealed that the water flows in a central direction in the valleys at the upper reaches of the water current (Fig. 7b, blue arrows), while water flows out of the circular structure in the valleys at the lower reaches of the water current (Fig. 7b, green arrows). There is little water flow in the valleys situated at the midpoint of the upper and lower reaches of the current. The direction of water flow on top of the peaks and the centre of the circular structure is the same as the current direction (Fig. 7b, deep blue arrows). The current speed in the central zone was reduced by 24.2% from that of outside the circular structure.