We described the external morphology of M. reevesii embryos at stages 13–24 and their gonadal structure at stages 16–24. Eye pigmentation was first clearly recognized by candling at stage 13; thus, our analysis began at stage 13. The key characteristics used for staging M. reevesii are summarized in Table 1. The eggs of M. reevesii at the time of arrival weighed between 7.72 and 15.8 g (mean 12.6 g) and remained nearly constant in weight throughout embryonic development (Fig. 1a, Table S1). The embryos grew faster at FPT than at MPT (Fig. 1b, Table S1) but reached similar body mass at each stage regardless of the incubation temperature (Fig. 1c, Table S1). No sexual dimorphic features were observed in respect of the external morphology during the embryonic development either at FPT (31°C) or MPT (26°C). The individuals representative of each stage are shown in Figures 2, 3, and 4 in lateral, ventral, and dorsal view, respectively. Histology sections of representative gonads for both FPT and MPT are shown in Figure 5.
Table 1
The key characteristics used for staging Mauremys reevesii.
Stage
|
Characteristics
|
13
|
Pigmented over retina
|
14
|
Pigmentation reaches pupil
|
15
|
Lateral margin of carapacial ridge is evident
|
16
|
Anterior end of carapacial ridge is evident
|
17
|
Posterior margin of plastron is evident
|
18
|
Digits II–IV protrude, and the thickness at the web is greater than the protrusion
|
19
|
Digits II–IV protrude as much as or more than the thickness at the web
|
20
|
Digits II–IV protrude approximately twice their thickness at the web
|
21
|
The scales on the dorsal forelimb are slightly visible, but do not reach the proximal border of the webbing
|
22
|
The scales on the dorsal forelimb extend to the ranges from the proximal border of the webbing to the distal region of the digits
|
23
|
The palmar surface of the forelimb is fully covered by small circular scales
|
24
|
The translucent sheath on the ungual phalanx recede toward its distal end
|
Embryonic development
Stage 13
In the lateral view, the anterior edge of the occipital protuberance extends anteriorly beyond the posterior edge of the eye. In the same view, the maxillary process encroaches anteriorly beyond the optic fissure. Pigmentation of the eye is dense, but the pupil and its margin are unpigmented. The optic fissure appears as an unpigmented “triangular” slit at the ventral region of the eye. The forelimb buds are longer than wide.
Stage 14
In the lateral view, the occipital protuberance recedes posteriorly as its anterior edge is near the level of the posterior edge of the eye. The area between the maxillary and lateral nasal processes—where the external nares are later formed—is weakly marked by shallow creases. The pigmentation of the iris reaches the margin of the unpigmented white pupil. The pharyngeal arches are evident. The anterior end of the mandible extends anteriorly beyond the level of the posterior edge of the eye. The forelimb bends in caudal orientation. The body is more flexed ventrally and the tail is longer and more curled than at stage 13. The genital protuberance is evident, with a crease at its posterior margin.
Stage 15
The pharyngeal arches disappear at this stage. The anterior end of the mandible extends to the level of the center of the eye. The carapacial ridge becomes visible in lateral view but its anterior edge remains absent and smooth. The digital plate is well formed. The posterior border of the genital protuberance is evident by a crease between the genital protuberance and the tail. The urogenital papilla is slightly distinguished from the entire genital protuberance by a shallow crease. The dorsal surface is slightly pigmented from the anterior edge of the carapace to the nostril and at the base of the tail.
Stage 16
The optic fissure is evident but becomes narrower, from a “triangular” to “teardrop” shape. An incipient tympanum is barely visible in lateral view. The digital plate has a smooth periphery. The carapacial ridge becomes thicker and its anterior edge is distinct in the lateral view. The pigmentation becomes denser on the dorsal surface of the neck and head regions and at the base of the tail.
Stage 17
The optic fissure is visible but becomes a narrow slit. The caruncle is visible as a small white mark on the rostral tip of the upper jaw. Each of the five digits can be distinguished by a ridge as the digital plate is slightly serrated. The posterior border of the plastron is evident. A crease on the posterior border of the genital protuberance extends more laterally. The urogenital papilla becomes larger and protrudes as it occupies a large proportion of the genital protuberance. Ribs can be slightly seen through the carapace in dorsal view.
Stage 18
The optical fissure disappears at this stage, and scleral papillae are evident. The caruncle is more evident. The mandible and the upper jaw make an almost complete closure as the mandible extends anteriorly to the level of the anterior end of the eyes in the lateral view. Digits II–IV protrude along the periphery of the digital plate but the thickness at the web is greater than the protrusion. Carapace pigmentation begins at this stage 18, such that the dorsolateral margins of the carapace are slightly pigmented, exposing the marginal scutes with faint borders. The plastron remains unpigmented, but it weakly shows the borders of the plastral scutes. The lateral sides of the tail are slightly pigmented, forming lines of pigments. A fold is evident surrounding the urogenital papilla.
Stage 19
The pigmentation on the head region becomes denser and the external nares are emphasized by the round, unpigmented areas. The lower eyelid is slightly formed. Scleral papillae are distinct. The mandible extends anteriorly, the ending of which is between the level of the anterior end of the eye and that of the nostril in the lateral view. The mandible and the upper jaw make a complete closure. Digits II–IV protrude as much as or more than the thickness at the web along the periphery of the digital plate. The carapace becomes more pigmented, evidencing the borders of all scutes. The three keels on the carapace are barely visible; one runs along the midline of the vertebral scutes and the other two run along the costal scutes.
Stage 20
The lower eyelid reaches the scleral papillae. The protrusion of digits II–IV proceeds and reaches approximately twice their thickness at the web. The digits are weakly pigmented. Rows of cutaneous papillae are slightly visible on the dorsal surface of the neck. The entire carapace is pigmented, but the vertebral scutes are more pigmented than the other scutes. The cutaneous papillae on the vertebral scutes are barely visible in some specimens. The urogenital papilla is evident and remains prolapsed from the vent.
Stage 21
The scleral papillae disappear or become unclear at this stage. The entire body becomes more evidently pigmented. The pigmentation of the digits becomes denser at the thinner distal ends than the thicker proximal regions on most of specimens, vaguely indicating the border of digits and claws. The scales are slightly visible on the dorsal forelimb, but do not reach the proximal border of the webbing. The cutaneous papillae on the vertebral scutes are evident. The urogenital papilla withdraws into the vent.
Stage 22
The iris becomes distinct and the lower eyelid reaches its outer margin. The ungual phalanx enclosed in a translucent sheath is clearly visible including its tip. Pigmentation becomes heavier on the dorsal forelimb. The scales on the dorsal forelimb extend distally to the ranges of the proximal border of the webbing to the distal region of the digits. The palmar surface of the forelimb is slightly covered by small circular scales, and in some specimens, these scales overlap adjacent scales. Four large circular scales are more evident and aligned along the anteroposterior axis of the palmar surface. Each of these four scales slightly overlaps or projects over the surrounding palmar surface. The claws are homogeneously opaque and white-colored with evident pigmentation. The carapace becomes darker so that the presence of cutaneous papillae on the entire carapace is emphasized. The intestinal loop is withdrawn into the body.
Stage 23
The ungual phalanx is evident in a translucent sheath. The palmar surface of the forelimb is fully covered by small circular scales, and their overlap to adjacent scales is evident. The density of the cutaneous papillae increased on the vertebral and pleural scutes. The plastron is more pigmented so that the whitish regions are less than the darker pigmented regions.
Stage 24
The embryos resemble the hatchlings. The translucent sheath on the ungual phalanx recedes toward its distal end, exposing the ventral surface of the ungual phalanx. The individual scales on the forelimb, hindlimb, and tail are evident and overlap one another. The pigmentation becomes darker throughout the body; the pigmentation of the carapace is dark brown in color but that of the plastron and skin is blackish brown. The color contrast increases on the head, exposing a discernible pattern on the lateral and ventral surface of the head.
Gonadal development
At stage 16, the gonadal ridges are present on the mesonephros of embryos. The gonads have an inner medulla consisting of sex cords and outer cortical layers with 1–2 cells in thickness. The germ cells, with large spherical nuclei, and the medullary sex cords become more evident at stage 17. At stage 18, the sex cords begin to degenerate in some specimens at FPT. By stage 20 at FPT, the sex cords become disorganized and the medullary region shows the homogeneous structures, while at MPT the sex cords become more distinct, developing into the seminiferous tubules. As the seminiferous tubules develop at stages 20 and 21, the germ cells are enclosed within the cords. During stages 20 and 21, the cortex becomes slightly enlarged and thicker at FPT; conversely, the cortex becomes a thin one-cell-thick layer at MPT. From stage 21, the tissue area connecting gonads and the mesonephros begins to decrease, with the gonads becoming merely connected by a narrow stalk of the connective tissues by stage 25. At stages 24 and 25 at FPT, the distinction between cortex and medullary region is more evident and the medullary region becomes vacuolated.