Fig 1.
External features of the S. stellaris egg case at stage 1.
The internal ellipsoid egg yolk and blastodisc visible at stage 1 of development. The egg case membrane was filled with jelly surrounding the yolk. There were four seawater slits located at each corner of the egg case, two can be seen from the front (as shown in Fig 1), and another two at the back of the egg case (not shown). The flat end of the egg case will be the site of opening during hatching, whereas the arched end remains firm and closed during hatching. There are four tendrils attached at each corner of the egg case. The key feature for stage 1 was no visible embryo on top of the egg yolk. See S1 File for original photographs of Fig 1 illustration.
Fig 2.
The inside of the S. stellaris egg case at stage 2.
A: The egg yolk mass and the associated embryo to scale. B: The magnified embryo with indication of key morphological features (lateral view). The embryo consists an anterior (head and trunk primordia) and posterior (tail bud), where the somites (segmented mesoderm) can be found. The key feature for stage 2 was the visible embryo developed on top of the egg yolk membrane without a long tail. See S2 File for original photographs of Fig 2 illustrations.
Fig 3.
The inside of the S. stellaris egg case at stage 3.
A: The ellipsoid egg yolk mass and the associated embryo to scale. B: The magnified embryo with indication of key morphological features (lateral view). C: The diamond-shape mouth opening (ventral view). The key feature for stage 3 was the growth of the long tail. See S3 File for original photographs of Fig 3 illustrations.
Fig 4.
The inside of the S. stellaris egg case at stage 4.
A: Six pairs of filament groups, and more apparent blood vessels on surface of spheroid external yolk sac (dorsal view). B: Developing yolk stalk, eye, and fins (lateral view). C: The magnified head and gill filaments (ventral view). The key feature for stage 4 was gill filament development. See S4 File for original photographs of Fig 4 illustrations.
Fig 5.
The inside of the S. stellaris egg case at stage 5.
A: Position of placoid denticles and caudal denticles (dorsal view). B: Body shape began to resemble an adult shark and the external yolk sac shrank visible in size (lateral view). C: The magnified head and reduced gill filaments (ventral view). The key feature for stage 5 was nearly complete loss of external gill filaments. See S5 File for original photographs of Fig 5 illustrations.
Fig 6.
The inside of the S. stellaris egg case at stage 6.
A: Position of placoid denticles, caudal denticles and banded patterns on the body (dorsal view). B: A pair of spiracles, five pairs of gill openings, and fins (lateral view). C: The magnified head (ventral view). The key feature for stage 6 was the loss of fin-folds. See S6 File for original photographs of Fig 6 illustrations.
Fig 7.
The inside of the S. stellaris egg case at stage 7.
A: Position of placoid denticles and banded patterns on the body (dorsal view). B: Fully developed eye and greater pigmentation on skin (lateral view). C: The magnified head and fully consumed yolk (ventral view). The key feature for stage 7 embryo was a fully consumed external yolk. See S7 File for original photographs of Fig 7 illustrations.
Fig 8.
7 key developmental stages of S. stellaris inside the egg case.
Stage 1 was characterized by no visible embryo. Stage 2 was identified as an early embryo formed on the surface of the egg yolk membrane. Stage 3 embryos had long tails and stage 4 embryos had externalized gill filaments. These filaments had shrunk by stage 5. In stage 6 the key feature was the loss of fin-folds, and in stage 7 the key feature was the fully consumed external yolk.
Table 1.
Comparison of 7 developmental stages inside the egg case from S. stellaris and S. canicula.
Fig 9.
S. stellaris body length and external yolk sac volume across the 7 developmental stages.
Total body length (cm, green, Y = 0.6416X − 1.674, R2 = 0.9898, r = 0.9949) and total external yolk sac volume (cm3, orange, Y = -0.7397X + 23.23, R2 = 0.9105, r = -0.9542) of S. stellaris embryo inside the egg case over development time (in weeks). Data are mean ± SD (n = 8). The beginning of each developmental stage is demarked with a vertical line.
Fig 10.
Average daily body length gain (ADL) during each of the 7 developmental stages.
A: ADL (cm day-1) of S. stellaris embryos during each developmental stage. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). B: ADL (cm day-1) of S. canicula embryos during each developmental stage. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). C: Comparison of ADL (% day-1) between S. stellaris (purple) and S. canicula (pink) embryos at each developmental stage. Values represent mean ± SD (n = 8). There was no statistically significant difference between species at any stage (Mann-Whitney U test, P < 0.05).
Fig 11.
Yolk consumption rate during the 7 developmental stages.
A: Yolk consumption rate (cm3 day-1) of S. stellaris embryos during the 7 developmental stages. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). B: Yolk consumption rate (cm3 day-1) of S. canicula embryos during the 7 developmental stages. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). C: Comparison of yolk consumption rate (% day-1) between S. stellaris (purple) and S. canicula (pink) embryos during 7 developmental stages. Values represent mean ± SD (n = 8). There was no statistically significant difference between species at any stage (Mann-Whitney U test, P < 0.05).
Fig 12.
Development time (age) to reach 7 developmental stages and hatch.
A: Development time (in weeks) for S. stellaris embryos to reach each developmental stage and hatch. Values represent mean ± SD (n = 8), whilst points show measurements from each individual. B: Development time (in weeks) for S. canicula embryos to reach each developmental stage and hatch. Values represent mean ± SD (n = 8), whilst points show measurements from each individual. C: Comparison of time spent in each developmental stage (as % of total developmental time) from lay to hatch for S. stellaris (purple) and S. canicula (pink). Values represent mean ± SD (n = 8). There was no statistically significant difference between species (Mann-Whitney U test, P < 0.05).
Fig 13.
Duration of development time of the 7 developmental stages.
A: Duration of development time (in weeks) spent in each of the 7 developmental stages for S. stellaris embryos. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). B: Duration of development time (in weeks) spent in each of the 7 developmental stages for S. canicula embryos. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). C: Comparison of duration of development time (as % of total time) between S. stellaris (purple) and S. canicula (pink) embryos at 7 developmental stages. Values represent mean ± SD (n = 8). There was no statistically significant difference between species (Mann-Whitney U test, P < 0.05).
Fig 14.
A: Embryo with curled body inside the egg case. B: Embryo facing the flat end of the egg case. C: Embryo tail pushed against arched wall of the egg case. D: Embryo pushed its body through the flat end of the egg case. E: Embryo swam out of the egg case.
Fig 15.
Shrinking rate of S. stellaris egg cases (% in area h-1) with and without jelly.
Values represent mean ± SD, while points show individual measurements. Asterisk indicates statistically significant effect of jelly on the rate of egg case shrinking (Mann-Whitney U test, P < 0.05).