Estimation and rapid identification of later stages during embryonic development of the oviparous lizard Sceloporus aeneus (Squamata: Phrynosomatidae)

Abstract

Stages of embryonic development for reptiles have been presented in tables that may include all or part of embryonic development. When oviposition occurs in some lizards, embryos are already in the later stages of development; likewise, the size of the eggs increases as incubation time progresses. However, it is unclear whether there is a relationship between the size of the egg and embryo development. Therefore, the objective of this study was to define whether there is a correlation between the size and volume of eggs and the embryonic stage of the lizard Sceloporus aeneus (Squamata: Phrynosomatidae). This study described the most relevant morphological changes in embryogenesis, from the moment of oviposition until shortly before hatching. Our results showed a statistically significant correlation between the stage of embryo development and the size and volume of eggs. Eleven development stages were characterized, initiating at the moment eggs were laid. Each developmental stage was mainly identified according to the morphological characteristics of the limbs and the body pigmentation. Our study links the growth of eggs with the developmental stage of embryos, which can provide a useful tool to those studies that require specific stages of the embryos and in those that undertake analysis of biological processes, such as organogenesis. The S. aeneus lizard manifests similar morphological and skeletal limb development to that documented for other species. Thus, analysis of limbs represents a quick and easy way to establish stages of embryonic development in S. aeneus, allowing us to compare their development with other species.

1. INTRODUCTION

Characterization of embryonic stages is extremely important for the study of evolutionary biology and embryology. A series of embryonic stages (st) has been described for some reptiles, which may fully or partially cover the development of the species of interest (Andrews, 2004; Wise et al., 2009). So far, embryonic development stages in turtles (Cordero & Janzen, 2014; Greenbaum & Carr, 2002; Magalhães et al., 2017; Werneburg et al., 2009; Yntema, 1968), crocodiles (Ferguson, 1985; Iungman et al., 2008; Peterka et al., 2010), snakes (Boback et al., 2012; Khannoon & Zahradnicek, 2017; Sandoval et al., 2020; Zehr, 1962) and lizards (Dufaure & Hubert, 1961; Griffing et al., 2019; Iungman et al., 2019; Lemus et al., 1981; Lin et al., 2021; Muthukkaruppan et al., 1970; Noro et al., 2009; Okuyama et al., 2021; Rapp Py‐Daniel et al., 2017; Sanger et al., 2008; Wise et al., 2009) have been described. Although the early development of embryos in squamate reptiles (Squamata: lizards and snakes) is apparently similar, descriptions of morphological characteristics of later development stages vary depending on the species analyzed or even the author descriptions (Boback et al., 2012; Cordero & Janzen, 2014; Peterka et al., 2010; Werneburg, 2009; Wise et al., 2009).

Each stage of development manifests specific morphological characteristics that gradually change as embryonic development proceeds. Among lizards, many descriptions of developmental stages have been based on those for Zootoca (Lacerta) vivipara by Dufaure & Hubert, 1961, who describe entire embryonic development divided into 40 stages; however, other developmental series have referred to distinctive features relating to 43 stages, including, for example, the typical pigmentation of the species (Griffing et al., 2019; Lin et al., 2021; Muthukkaruppan et al., 1970; Okuyama et al., 2021; Wise et al., 2009). Some tables, such as those presenting the developmental stages of the gecko Paroedura pictus, refer to the days elapsed from the moment of oviposition, and are based exclusively on the external morphology (Noro et al., 2009). Tables of normal development are based on distinctive morphological characteristics present at each stage of development. Each stage of embryonic development is defined by various characteristics, including embryo size, number of somites, facial prominences, neural tube vesicles, pharyngeal arches, eyes, limbs, formation of internal organs, and pigmentation. Many of these characteristics cannot be directly evaluated when the embryos are obtained; therefore, careful observations and measurements are required to confirm development stages (Werneburg, 2009).

Among squamate reptiles, most species are oviparous and exhibit uterine egg retention (Andrews & Mathies, 2000; Blackburn, 2000). In many lizards, early developmental stages such as blastula cleavage and gastrulation occur in the reproductive tract, so when oviposition occurs, embryos are at advanced stages of development (Shine, 1983). Additionally, it has been observed that oviposition may occur within a range between st26 and st33 of the embryonic stages (Andrews & Mathies, 2000; Shine, 1983). In particular, species of the genus Sceloporus manifest widely varied oviposition periods, extending from st27 to st39 (de Marco, 1993; Calderón‐Espinosa et al., 2006; García‐Collazo et al., 2012). The developmental series in oviparous lizards are usually partial and cover the post‐ovipositional developmental process, which includes the organogenesis period and body size growth (Noro et al., 2009; Okuyama et al., 2021; Sanger et al., 2008; Wise et al., 2009).

Among lizards, it has been observed that following oviposition, eggs undergo modifications such as change of shape or increase in size (width, length), volume (mm³) and mass (mg). Some of these changes have been observed in the lizards Liolaemus tenuis tenuis (Lemus et al., 1981; 1984), Podarcis muralis (Ji & Braña, 1999) and Sceloporus aeneus (Guillette, 1981). The eggs of the P. muralis lizard have a flexible type of shell, and during embryonic development, the egg mass was seen to increase due to water absorption (Ji & Braña, 1999).

Eggs from S. aeneus have thin and highly extensible shells (Guillette & Jones, 1985) and their size and shape have been observed to change during their incubation period (Guillette, 1981); however, the relationship between egg size and volume with stage of embryonic development has not been analyzed. Therefore, in this study, we aimed to correlate the size and volume of eggs with embryonic stages and describe the most relevant morphological changes during embryogenesis (from the moment of oviposition until shortly before hatching) of the oviparous lizard S. aeneus (Squamata: Phrynosomatidae) in order to rapidly identify post‐oviposition stages.

2. MATERIALS AND METHODS

2.1. Animal collection and conservation status

The Sceloporus aeneus lizard (Wiegmann, 1828) is endemic to México and in the category of least concern (LC) on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List (Canseco‐Márquez et al., 2007). Fourteen gravid females from the lizard S. aeneus (Figure 1a) were collected in Milpa Alta (19°11′53″ N, 99°50′49″ W), a suburb of southwest México City during the month of June 2012 (Scientific collector permit: FAUT 0186 SEMARNAT). Animals were kept in plastic cages with natural substrate and water, and food consisting of flies and crickets. All females were marked with random serial numbers and monitored until oviposition. Numbers were marked on the dorsal region of the body using a pencil with non‐toxic white paint. This allowed us to relate each nest obtained with the corresponding female. Each nest was placed in an individual thermal container covered with natural substrate and maintained at room temperature to be incubated under the same conditions. Thermal containers were identified with the following data: number of the female that laid eggs, date of oviposition, and number of eggs. Each egg was marked with a graphite pencil to identify the corresponding female/nest number and a random consecutive number using points.

FIGURE 1.

Sceloporus aeneus female and egg size. Image showing an adult female of the S. aeneus species collected in Milpa Alta, México (a). Representative sample of eggs collected from several nests where different egg sizes are observed (b). Each egg was identified with a consecutive number and the key to corresponding nest (numbers and points). Eggs obtained at different times of incubation showing their approximate sizes using a millimeter ruler (c)–(e). Embryos obtained from the eggs shown in the corresponding upper images (f)–(h). Embryos are shown associated with the yellow yolk. Image of an egg obtained at the time of oviposition (c) containing an embryo at st30 (f). Representative image of an egg during incubation period (d) containing an embryo at st35 (g). Greater size egg (e) collected at the end of embryonic development at st40 (h).

2.2. Embryo collection and obtaining biological samples

All laboratory procedures were undertaken following ethical norms for animal experimentation as directed by the Ethics and Scientific Responsibility Commission of the Facultad de Ciencias, Universidad Nacional Autónoma de México and in compliance with the Guidelines for the Use of Animals (2012). Eggs were randomly extracted from each nest, and embryos were photographed fresh and then sacrificed by decapitation. Heads and bodies of embryos were fixed in 4% paraformaldehyde (PFA, Sigma‐Aldrich, St Louis, MO, USA) in phosphate‐buffered saline (PBS, pH 7.1, Gibco, Grand Island, NY, USA) and dissected limbs were fixed in 70% ethanol (JT Baker) in bi‐distilled water for further morphological analysis.

2.3. Developmental stages

The length and width of each egg were measured using a Vernier caliper, and the number of incubation days at the time of slaughter was noted. Embryo developmental stages were identified after comparing with the table presenting stages of embryonic development of the European lizard Z. vivipara (Dufaure & Hubert, 1961) and confirmed by analyzing the morphological characteristics of the limbs and the head of each specimen. This table was used because it includes descriptions of the entire embryonic development, and late stages can be identified based on limb morphology. Morphological analysis was performed using a stereo microscope (Zeiss, Stemi 2000‐C) and a digital camera (Sony Cyber‐Shot, DSC‐S75) for image capture.

Stages identified in S. aeneus were compared with the embryonic development of other recently studied lizards. Additionally, a comparison was made with the embryonic development of the laboratory mouse Mus musculus. To carry out the correlation with mouse, morphogenesis of the limbs was considered the main characteristic of embryonic development.

2.4. Staining of limbs

In order to study the skeletal development of limbs, all samples were fixed in 70% ethanol (Sigma‐Aldrich, USA) and double stained for cartilage (Alcian blue) and bone (Alizarin red). Limbs were dehydrated in ethanol 100% for 24 h and permeabilized in acetone for 24 h. Samples were incubated at 37°C for 24 h in a staining solution, which consisted of 0.005% Alizarin red, 0.015% Alcian blue, 60% Ethanol, and 0.05% Acetic acid. Subsequently, samples were washed with bi‐distilled water and then cleared with a 1% KOH/20% glycerol solution for 24 h. Finally, limbs were stored in a 1:1 solution of glycerol/70% ethanol. Photographs were taken with a digital camera (Sony Cyber‐Shot, DSC‐S75) coupled to a stereo microscope (Zeiss, Stemi 2000‐C).

2.5. Statistical analysis

The volume of each egg was obtained, applying the following equation: V = 4/3πa ² b. Where π = 3.1416; a ² = (0.5 width)²; b = (0.5 length). The values for egg length (mm), width (mm) and volume (mm³) were presented as mean ± standard deviation, referring to a total of 90 eggs collected randomly. In order to determine the relationship between developmental stages, length, egg volume, or width of embryos, an analysis of variance (ANOVA) and analysis of correlation for continuous and independent data were performed using the Statgraphics Centurion XV.I (Statgraphics Technologies, Inc.). Statistical significance was evaluated with p‐values less than 0.05.

3. RESULTS

3.1. Egg size and volume

Pregnant females were kept in captivity until oviposition; the captivity period lasted 28 days from the first to the last oviposition. Because of this, at the moment of collection, eggs in different nests varied in size (Figure 1b). Eggs are oval‐shaped and have a smooth, shiny white shell throughout the incubation period. When embryos fail to develop and die, the eggs have an irregular shape and opaque spots; therefore, eggs with these characteristics were not included in this study. At the moment of oviposition, eggs were small, and dissection of the embryos showed that they usually were at st30 of the development (Figure 1c, f). Likewise, later stages of embryonic development correlate with an increase in the size of eggs; medium‐sized eggs were usually observed when embryos were at st35 (Figure 1d, g) and larger eggs corresponded to the st40 (Figure 1e, h). Length, width, and egg volume increased as incubation time progressed. The ANOVA values for the length of the eggs were F (10, 58) = 39.03 (p <0.05) and for the width were F (10, 58) = 29.60 (p <0.05). These results showed a statistically significant difference between the means of the 11 developmental stages analyzed at the 95.0% confidence level.

Table 1 shows the average size and egg volume of S. aeneus eggs for each embryo stage identified. Correlation analysis showed that there is a statistically significant relationship between the stage of embryo development with respect to size (length or width) and the volume of eggs. The developmental stage variable manifests values for the correlation coefficient of 0.93 referring to length, 0.91 for width, and 0.90 for volume (p <0.05).

TABLE 1.

Average size and volume of the eggs from oviparous lizard Sceloporus aeneus.

Stage	n	Incubation days	Length (mm) a	Width (mm) a	Volume (mm3) a
st30	6	0	7.9 ± 0.2	5.7 ± 0.4	132.6 ± 20.9
st31	2	2	8.4 ± 0.1	6.0 ± 0.1	155.8 ± 6.3
st32	10	5	9.2 ± 0.7	6.8 ± 0.3	221.6 ± 33.4
st33	13	10	9.5 ± 0.5	6.8 ± 0.5	230.5 ± 46.4
st34	5	10–11	10.0 ± 1.0	7.7 ± 0.9	346.4 ± 83.0
st35	12	11–17	12.2 ± 0.7	9.7 ± 0.6	612.2 ± 95.6
st36	11	15–25	12.3 ± 0.5	9.6 ± 0.9	599.3 ± 146.7
st37	8	21–28	12.7 ± 0.8	10.0 ± 0.5	671.3 ± 85.2
st38	8	27–32	13.4 ± 1.1	10.0 ± 1.1	717.8 ± 191.7
st39	8	32	13.8 ± 0.4	10.5 ± 0.5	801.8 ± 89.6
st40	7	42–47	14.7 ± 0.7	12.2 ± 0.6	1145.6 ± 145.9
Coefficient of correlation			0.93	0.91	0.90

^a Average values ± standard deviation of the length, width (mm) and egg volume (mm³) of S. aeneus in each of the stages of embryonic development analyzed. Coefficient of correlation was obtained with a p <0.05.

3.2. Developmental stages of S. aeneus embryos

Embryos of S. aeneus were collected from the moment of oviposition until shortly before hatching. 11 embryonic stages (st30–st40) were identified, based on the table for the European lizard Z. vivipara (Dufaure & Hubert, 1961). Table 2 shows the correlation of embryonic development of S. aeneus, with the most recent studied lizards and the laboratory mouse. Each stage was recognized mainly in terms of limb formation and other morphological characteristics acquired by the embryo during its development. Representative images of S. aeneus embryonic development from st30 to st40 are shown in Figures 2, 3, 4. The embryological stages and the main characteristics are described below.

TABLE 2.

Comparison of embryonic development between Sceloporus aeneus, recently studied lizards, and mouse.

Sceloporus aeneus	Eublepharis macularius	Eremias multiocellata	Lepidodactylus lugubris	Salvator merianae	Takydromus tachydromoides	Tarentola annularis	Tropidurus torquatus	Varanus indicus	Mus musculus
st30	st30	st29	st30	st8, D10	st29	st29	st30	10 dpo	Lst 3
st31	st31	st30‐st31	st31	st8, D10	st30	st30‐st31	st30‐st32	‐	Lst 4–5
st32	st32	st32	st32	st9, D11‐13	st31	st31‐st32	st33	24 dpo	Lst 6–7
st33	st33	st33	st33	st10, D14‐15	st32‐st33	st32‐st33	st33	32 dpo 43 dpo	Lst 8
st34	st33	st33	st34	st11, D18	st34	st33	st34	56 dpo	Lst 9
st35	st34‐st35	st34	st35	st12, D21 st13, D24	st34‐st35	st34‐st35	st34‐st35	63 dpo	Lst 10–11
st36	st35‐st36	st35‐st36	st36‐st37	st13, D24 st14, D27	st35‐st36	st36‐st37	st35‐st36	73 dpo	Lst 12
st37	st36‐st37	st36‐st37	st37‐st38	st14, D27	st36‐st37	st37‐st38	st36‐st37	73 dpo 81 dpo	Lst 13–14
st38	st38‐st39	st38	st38‐st39	st14, D27	st37‐st38	st38	st38‐st39	88 dpo	Lst 15
st39	st40‐st41	st39	st39‐st40	st14, D30 st15, D33	st39	st38‐st39	st40‐st41	95 dpo 103 dpo	Lst 15
st40	st41‐st42	st40‐st42	st40‐st42	st16, D42 st17, D47–48 st18, D51–57	st40‐st42	st39	st41‐st42	130 dpo 160 dpo	Lst 15

Note: Comparison of the embryonic development of lizards Sceloporus aeneus (this study) Eublepharis macularius (Wise et al., 2009), Eremias multiocellata (Lin et al., 2021), Lepidodactylus lugubris (Griffing et al., 2019), Salvator merianae (Iungman et al., 2019), Takydromus tachydromoides (Okuyama et al., 2021), Tarentola annularis (Khannoon, 2015), Tropidurus torquatus (Rapp Py‐Daniel et al., 2017), Varanus indicus (Gregorovicova et al., 2012) and mouse Mus musculus (Wanek et al., 1989). The classification criteria are based on general morphological characteristics of the embryos. S. aeneus embryos were classified according to the morphological characteristics of the limbs described in Zootoca vivipara (Dufaure & Hubert, 1961). D, days of incubation; dpo, days post oviposition; Lst, limb development stage; st, embryonic development stage.

FIGURE 2.

Developmental stages of Sceloporus aeneus embryos. Representative images show the morphological characteristics of S. aeneus embryos at st30 (a), st31 (b) and st32 (c). Specific characteristics and limbs corresponding to each stage of development are shown in images located on the right side of each embryo. In the amplifications, forelimbs are shown on the left side and hindlimbs on the right side. The double‐headed arrow indicates direction of the proximal‐distal axis (prox‐dis) of both limbs. Ap, autopodium; ES, endolymphatic sac; E, eye; FL, forelimb; Fnp, fronto‐nasal processes; H, heart; HL, hindlimb; Li, liver; Mnp, mandibular process; Mxp, maxillary process; Mes, mesencephalon; MK, mesonephric kidney; P, pupil; Sm, somites; T, tail; Zp, zeugopodium. Bar = 2 mm.

FIGURE 3.

Developmental stages of Sceloporus aeneus embryos. Representative images show the morphological characteristics of S. aeneus embryos at st33 (a), st34 (b) st35 (c)and st36 (d). Specific characteristics and limbs corresponding to each stage of development are shown in images located on the right side of each embryo. In the amplifications, forelimbs are shown on the left side and hindlimbs on the right side. Ap, autopodium; DC, digital condensation; D, digits; ES, endolymphatic sac; E, eye; El, eyelid; FL, forelimb; HL, hindlimb; Hp, hemipenes; IM, interdigital membrane; I, iris; Mes, mesencephalon; Mn, mandible; Mnp, mandibular process; Mx, maxilla; Mxp, maxillary process; P, pupil; PO, pineal organ; PS, pigmented spots; RS, ring of sclera; Sp, stylopodium; T, tail; Tel, telencephalon; Zp, zeugopodium. Bar = 2 mm.

FIGURE 4.

Developmental stages of Sceloporus aeneus embryos. Representative images show the morphological characteristics of S. aeneus embryos at st37 (a), st38 (b), st39 (c), and st40 (d). Specific characteristics and limbs corresponding to each stage of development are shown in the amplifications located on the right side of each embryo. In the amplifications, forelimbs are shown on the left side and the hindlimbs on the right side. B, brain; El, eyelid; Hp, hemipenes; N, nails; S, scales; SCS, stripes with clear scales. Bar = 2 mm.

st 30. The cephalic neural tube is divided into five vesicles; the mesencephalon is prominent. The eyes are large, well defined, and slightly pigmented in the choroid membrane. The developing heart, liver, mesonephric kidney, and other organs are visible. The tail has been formed. The four limb buds are round in shape, and visible at both sides of the main axis of the embryo. The eggs measured 7.9 ± 0.2 mm length by 5.7 ± 0.4 mm width (Figure 2a).

st 31. In the occipital region, near to the mesencephalon, two small white spots are visible; these are endolymphatic sacs. The eyes are bulbous and slightly more pigmented at the border and around the pupil region. The front‐nasal processes can be distinguished in the facial region. Limb buds have flattened ends, and the hindlimb is slightly longer than the forelimb. The eggs measured 8.4 ± 0.1 mm in length by 6.0 ± 0.1 mm in width (Figure 2b).

st 32. The mesencephalon begins to form two lobules. The white endolymphatic sacs become more evident. The eyes are kidney‐shaped; the pupil is well defined by dark pigment. The maxillary process is fused to the front‐nasal process. The mandibular process begins to differentiate. Autopodium and zeugopodium are distinguishable; the autopodium is similar to a palm. The eggs measured 9.2 ± 0.7 mm in length by 6.8 ± 0.3 mm in width (Figure 2c).

st 33. The mesencephalon is divided into two elongated lobules. The eyes present a longitudinal ring in the sclera. The autopodium, zeugopodium, and stylopodium of the limbs are differentiated. The ends of the palms are wavy, and chondrogenic condensations are visible in the digital region. The hemipenes begin to differentiate and look like little buds. The eggs measured 9.5 ± 0.5 mm in length by 6.8 ± 0.5 mm in width (Figure 3a).

st 34. The lobules of the mesencephalon are lateral and spherically shaped. The telencephalon is more prominent. The endolymphatic sacs are fused. The inner and outer edges of the iris are well defined. The mandible is shorter than the maxilla; approximately one‐half to three‐quarters of the maxilla. Hindlimbs are slightly longer than forelimbs; autopodium has been differentiated into five digits, joined together by an interdigital membrane. Hemipenes are more evident. The eggs measured 10.0 ± 1.0 mm length by 7.7 ± 0.9 mm width (Figure 3b).

st 35. The lobules of mesencephalon and telencephalon are closely related in the upper region of the head. The pineal organ is pigmented and is in the telencephalon region. The eyes are located more frontally in the face. The mandible is slightly shorter than the maxilla. The digits are totally formed, partially joined, and there is evident regression of interdigital tissue. The eggs measured 12.2 ± 0.7 mm length by 9.7 ± 0.6 mm width (Figure 3c).

st 36. The upper and lower eyelids are opaque and partially cover the eyes. The pupil and iris are well defined in the central region of the eyes. The maxilla and mandible are similar in length. The digits are slightly annulated and completely separate; pigmented spots are visible in the region of the nails. The fourth digit is longer than the other digits. Scales are slightly delimited on the dorsal region of the body; some pigmented spots are visible. The eggs measured 12.3 ± 0.5 mm in length by 9.6 ± 0.9 mm in width (Figure 3d).

st 37. The eyelids form an internal oval border and cover the eye to the outer edge of the pupils. The embryo has pigmented scales in the dorsal region. Scales are delimited and not pigmented on the stylopodium. The nails are well demarcated and pigmented. The eggs measured 12.7 ± 0.8 mm length by 10.0 ± 0.5 mm width (Figure 4a).

st 38. The eyes are covered almost entirely by the upper and lower eyelids. The body is covered with pigmented scales except on the head and ventral region. The pattern of pigmentation, specific to this species, begins to be evident. The limbs are partially covered with pigmented scales. Nails are well differentiated; digits are covered, and scales lack pigment. The eggs measured 13.4 ± 1.1 mm in length by 10.0 ± 1.1 mm in width (Figure 4b).

st 39. The brain is visible because of the absence of scales on the head. Face and eyelids are covered with slightly pigmented scales, whereas the body has more pigment. Limbs and digits are completely covered by pigmented scales. Hemipenes are larger among males. The eggs measured 13.8 ± 0.4 mm length by 10.5 ± 0.5 mm width (Figure 4c).

st 40. The entire embryo is covered with scales and has acquired typical dark pigmentation of the species. Two pairs of stripes with clear scales are visible on the lateral region of the body. Upper and lower eyelid membranes are closed, and the eyes are not more visible. Hemipenes are visible in male embryos. The eggs measured 14.7 ± 0.7 mm in length by 12.2 ± 0.6 mm in width (Figure 4d).

3.3. Limb ossification among S. aeneus embryos

The limbs of the S. aeneus embryos were fixed and stained with Alcian Blue and Alizarin Red to reveal the development of cartilage and bone, marked in blue and red, respectively. From st30 to st32, limbs are formed from undifferentiated mesenchyme, and neither cartilage nor bone was detected (data not shown). From st33 to st39, the formation and development of cartilage is observed in the main skeletal structure of the limbs. The beginning of the ossification process was evidenced by scarce blue staining in the central region of the long bones (humerus, ulna, radius, femur, tibia, fibula) at st36 to st39. Complete ossification in the limbs of S. aeneus is observed at st40, when red staining was evident in the long bones and phalanges. Figure 5 shows representative images of skeletal development in the limbs of S. aeneus embryos at st33, st34, st36, st38, and st40. The main characteristics of skeletal development are described below.

FIGURE 5.

Limb ossification of the Sceloporus aeneus. Representative limbs of S. aeneus cleared and stained with Alcian Blue and Alizarin Red. Forelimbs (FL) and hindlimbs (HL) obtained at st33 (a), st34 (b), st36 (c), st38 (d) and st40 (e). Light blue stain is observed in the central region of the long bones (arrowhead) and in the connective tissue. Dark blue stain is detected in primary skeletal elements (a)–(d); as well as in the epiphysis and carpal and tarsal elements (e, white arrows). Red stain is observed in calcified structures (e, asterisk). The carpal and tarsal elements at each stage are shown in right‐side amplifications (b)‐(e). On both limbs, five digits (I–V) are observed with a phalangeal formula of 2‐3‐4‐5‐3 for forelimbs and 2‐3‐4‐5‐4 for hindlimbs. Forelimbs: H, humerus; R, radius; U, ulna; Ca, carpals; Mc, metacarpals; Ph, phalanges. Hindlimbs: Fe, femur; F, fibula; T, tibia, Ta, tarsals; Mt., metatarsals; Ph, phalanges (Ph). Carpal elements: Ul, ulnare; Ra, radiale; CC, carpal centrale; DC 1–5, distal carpal 1–5. Tarsal elements: Fi, fibulare; IC, intermedium centrale; DT 2–4, distal tarsal 2–4, F/ic, fibulare and intermedium‐centrale fused; PT, proximal tarsal. Bar = 2 mm.

st33. Cartilage formation was evident by Alcian Blue staining; formation of the long bones of the stylopodium and zeugopodium is slightly visible. In the forelimb, the humerus is visible in the stylopodium, and the ulna and radius are observed in the region of the zeugopodium. The femur is visible in the stylopodium of the hindlimb, and the fibula and tibia are visible in the zeugopodium region. In the autopodium region of both limbs, the primary axis of metacarpals and metatarsals is slightly defined (Figure 5a).

st34. At this stage, the humerus, ulna, radius, femur, fibula, and tibia are completely formed by cartilage. Skeletal tissue of carpals and metacarpals is apparent in the autopodium region of the forelimbs, whereas the tarsals and metatarsals bones are formed in the autopodium region of the hindlimbs. Five metacarpals and five metatarsals are visible in the limbs. Seven carpal elements are visible: ulnare, radiale, centrale, and four distal carpals. Four tarsal elements are visible: fibula, intermedium‐centrale, and two distal tarsals. The primary axis of all five digits is well defined; phalanges begin to develop at both limbs (Figure 5b).

st36. Development of all phalanges at both limbs is visible from st35. Formula for the phalanges is 2‐3‐4‐5‐3 for forelimbs and 2‐3‐4‐5‐4 for hindlimbs. Distal carpal 1 is slightly visible in the forelimbs. Fibula and intermedium‐centrale elements are fused, and the distal tarsal 2 is visible in the hindlimbs. Scarce blue‐staining in the central region of long bones, metacarpals, and metatarsals is visible (Figure 5c).

st38. The scarce blue‐staining in the central region of phalanges is also evident. At this stage, distal carpal 1 and distal tarsal 2 are not visible (Figure 5d).

st40. The ossification process has taken place; the blue‐stained cartilage is detected at the ends (epiphysis) of bones that appear stained red. All carpal elements, distal tarsal, and joints are stained in blue; the proximal tarsal is ossified (Figure 5e).

4. DISCUSSION

Among reptiles, three types of eggshells are found: (1) soft or extremely flexible shells, (2) semi‐rigid or flexible shells, and (3) hard or rigid shells (Legendre et al., 2022; Packard et al., 1982; Packard & de Marco, 1991). Eggs of S. aeneus and other Sceloporus species present extremely flexible and thin eggshells, making it possible for eggs to increase in size (Andrews, 1997; Guillette, 1981; Guillette & Jones, 1985). In the present study, we found that the increase in size of eggs from S. aeneus correlates with later stages of embryo development, in a way similar to other lizards such as P. muralis (Ji & Braña, 1999). The smallest embryos at st30 were observed at the moment of oviposition, whereas the largest embryos at st40 were present at stages close to hatching. The increase in egg size indicates that embryonic development is proceeding properly. In controlled laboratory conditions, egg incubation days provide information on the progress of embryonic development from the date of oviposition (Noro et al., 2009). However, when eggs are obtained from nests under natural conditions and transported for laboratory maintenance, incubation days reflect a lagged developmental progress. Therefore, the relationship between egg size and embryonic development allows us to estimate the progress of development in nests where the date of oviposition is unknown.

Eggs with rigid shells can be opened, micro‐manipulated, and subsequently incubated (Noro et al., 2009); however, manipulation is limited or not possible for flexible and extremely flexible eggshells. This fragility of eggshells makes it difficult to obtain embryos at specific stages of development. For S. aeneus, it was evident that average values of egg size and volume are related to different stages of embryonic development. Therefore, by quantifying these variables during the incubation period, it is possible to estimate the embryonic stage. In this study, we propose a quantitative method to estimate the embryonic stages of S. aeneus that may be useful when these are collected for analysis. This makes it easier to identify specific stages, thus reducing the number of embryos necessary for experimental research. Development of various tissues and organs is widely studied in mammals but is less known in reptiles and other vertebrates. Previous work in lizards has focused on the study of gonadal development (Antonio‐Rubio et al., 2015; Doddamani, 2006; Neaves et al., 2006; Rams‐Pociecha et al., 2024). It has been reported that in S. aeneus embryos the undifferentiated gonad is observed between st30 to st33 and the ovaries or testes from st34 onwards (Antonio‐Rubio et al., 2015). Here we report that egg size allows us to calculate the stage of embryo development. In this way, targeted analyses can be performed at specific stages where structures of interest are found, such as the differentiating gonads. Therefore, for studies of sex determination and gonadal differentiation in S. aeneus, it is necessary to focus on small eggs in which embryonic development is in st30 to st34. Furthermore, this type of research can be extrapolated to other species of lizards that have eggs with flexible shells; for example, some species of the genera Anolis or Iguana (reviewed in Packard et al., 1982).

Eggs with extremely flexible shells are sensitive to changes in the environmental conditions where they are incubated. Therefore, it is important to consider other variables such as humidity, temperature, and time of incubation (Ji & Braña, 1999). This type of eggshell allows the water to filter into the egg; therefore, variations in the hydric environment may act as a limiting factor when attempting to define developmental stages in terms of size. Similarly, in the lizard Callisaurus draconoides, egg mass increases or decreases in relation to absorption or loss of water, respectively (Packard et al., 1980). It has been suggested that favorable water conditions during incubation have a positive effect on the quality of hatchlings (Packard et al., 1982) and it has been proposed that water can influence the amount of yolk that is consumed during embryonic development (Booth & Yu, 2009). It is thus important to maintain good moisture conditions for eggs during the incubation period. S. aeneus eggs have been reported to measure 4.2 mm long by 3.4 mm wide at the moment of oviposition and then 10.3 mm long by 6.75 mm wide prior to hatching (Guillette, 1981). However, our data indicated that S. aeneus eggs from Milpa Alta, México City, have a greater average size compared to other locations; thus, the location where eggs are collected may also represent another important variable.

It has been observed that the temperature of incubation affects the growth and development of embryos, especially during their early stages, and that a variation in rate may occur among embryos of the same age (Andrews, 2004). Therefore, a categorization of embryos based on days of incubation may not be indicative. Apparently, embryo development among squamous reptiles (Squamata) is similar; however, evidently among these species, certain morphological characteristics appear at slightly different moments during development (Neaves et al., 2006; Sanger et al., 2008; Wise et al., 2009). In this sense, Wise et al. (2009) and Khannoon (2015) developed a comparative table that presented stages of development in lizards, showing that embryos with similar morphological characteristics may be classified at different stages of development. This variation in embryo classification was also observed among lizard species included in the comparative table of this work. In relation to these observations, we suggest that these differences may be due to one or more of the following reasons: (1) the table used to establish the stages, (2) total number of stages which can be from 40 to 44, (3) classification based on days of incubation, (4) classification based on morphological characteristics, and (5) description of particular characteristics of each species.

A guide has been developed which establishes 104 characteristics that are common to all vertebrates and useful for describing stages of embryonic development of any vertebrate species (Werneburg, 2009). However, the analysis of each of these characteristics is generally carried out on fixed samples to provide more detail; fresh sample analysis is rare. Lizard S. aeneus is an oviparous species that undergoes egg retention (García‐Collazo et al., 2012; Guillette, 1981, 1982; Guillette & Lara‐Gongora, 1986), so the early stages of embryonic development (st1–st29) are carried in the reproductive tract and up until now, have not been analyzed. In this study, 11 stages of development were described in S. aeneus (st30–st40), which range from the moment of oviposition until shortly before hatching. In some tables, such as the table described for several species from the Anolis genus, advanced stages of embryonic development are included, ranging from shortly before oviposition until hatching (Sanger et al., 2008). These stages are similar to those described in the present study for S. aeneus; however, the table for Anolis describes important morphological characteristics in the biology of the genus, which may not be identified in species from other genera. Other tables present more than 40 stages of development, with specific patterns of color described for the final stages of the studied species (Griffing et al., 2019; Lin et al., 2021; Muthukkaruppan et al., 1970; Okuyama et al., 2021; Wise et al., 2009).

In S. aeneus, we can predict the stage of embryonic development by the egg size and days of incubation; however, embryos must be properly classified by analyzing their morphological characteristics. The present study describes the morphological characteristics that facilitate identifying the stage of development when fresh embryos are obtained. These stages of development were established based on the table presented by Dufaure and Hubert (1961), which has been widely used in the embryonic study of many lizards because it includes stages from segmentation to hatching, and late stages can be established according to the development of the limbs. During organogenesis, limbs are obvious structures that change morphologically as embryonic development progresses. According to Hopwood (2007), rapid categorization of study specimens in an experiment should be possible based on external characteristics defined using shape‐changing characters, and limb development is a good parameter. In this sense, we believe that a complement based on limb development could address the limitations of current staging tables. Studies carried out on oviparous species have considered the formation of limbs, digits, and nails as important features that characterize later stages of development (Khannoon, 2015; Lemus et al., 1981; Muthukkaruppan et al., 1970; Noro et al., 2009; Sanger et al., 2008; Wise et al., 2009). Limb development is a constant indicator in relation to embryo morphogenesis, so in this study we also considered the development of limbs as the main indicator defining later stages in the development of the S. aeneus lizard. Likewise, the limbs can be analyzed at the time of collection, which makes identification of the developmental stage quick and easy. This prevents the biological samples of interest from degrading or altering because of necrotic processes. Besides this, it is also important to consider that the number of digits in fore or hind limbs, scales as well as other features appear at stage‐specific levels; combining these with general characteristics may define a specific group of reptiles.

First recognized by Ernst Haeckel, heterochrony is defined as “change to the timing or rate of developmental events, relative to the same events in the ancestor” (reviewed in Dobreva et al., 2022; McNamara, 2012). Bininda‐Emonds et al. (2007) have reported heterochronic changes during limb development in several tetrapods, showing that in some amphibians, such as Xenopus leavis and Eleutherodactylus coqui, the hindlimb development precedes to the forelimb development. Meanwhile, in marsupial mammals, forelimbs develop earlier than the hindlimb (Bininda‐Emonds et al., 2007). Regarding reptiles, synchronous development was considered in Lacerta viridis and Emys orbiculare because no statistically significant heterochrony was observed between the development of the hind and forelimbs. In S. aeneus, we can infer that developmental pattern appears to be synchronous, because the external morphology and skeletal development were observed to be similar between the forelimbs and hindlimbs. However, a detailed analysis is needed to provide quantifiable data that can be compared with previous work.

Although there are various studies about limb development in reptiles (Burke & Alberch, 1985; Cordero and Janzen, 2014; Fabrezi et al., 2007; Leal et al., 2010; Mathur & Goel, 1976; Mohammed, 1984; Noro et al., 2009; Roscito et al., 2014; Sánchez‐Villagra et al., 2007; Sanger et al., 2012; Sheil & Portik, 2008), limb morphogenesis is generally considered a similar process to other vertebrates such as birds (Hamburger & Hamilton, 1951) and mammals (Wanek et al., 1989). It is important to highlight that studies in squamate reptiles cover a wide variety of taxa (Fabrezi et al., 2007; Leal et al., 2010; Mathur & Goel, 1976; Mohammed, 1984; Noro et al., 2009; Roscito et al., 2014; Sanger et al., 2012), and from our knowledge, this work is the first report describing morpho‐skeletal characteristics of limb development in a phrynosomatid lizard. Limb development begins with the formation of small lateral bud primordia, which increase in length, and mesenchymal cells differentiate to establish three major regions according to a specific pattern: autopodium, zeugopodium, and stylopodium. In each of these regions, specific bone structures are differentiated together, which form the entire limb. This general pattern of morphological limb development is similar to that observed in S. aeneus and other reptiles such as turtles Chelonia mydas, Caretta caretta (Sánchez‐Villagra et al., 2007), Emys orbicularis (Bininda‐Emonds et al., 2007), Trachemys scripta (Sheil & Portik, 2008) and Chrysemys picta (Cordero & Janzen, 2014), lizards Calotes versicolor (Mathur & Goel, 1976), Chalcides ocellatus (Mohammed, 1984) and Lacerta viridis (Bininda‐Emonds et al., 2007), the gecko P. pictus (Noro et al., 2009), several species of Anolis genus (Sanger et al., 2012) and studied species of amphibians, birds, and mammals (Bininda‐Emonds et al., 2007).

It has been proposed that limb development can be divided into three phases that encompass various stages of embryonic development: (1) limb bud outgrowth and patterning, (2) morphogenesis, and (3) growth (Sanger et al., 2012). During limb bud outgrowth and patterning, the limbs constitute undifferentiated mesenchyme, which we identified in S. aeneus at st30–st32. During this phase, the stylopodium, zeugopodium, and autopodium regions of limbs are delimited, and no developed skeletal elements were observed. During the morphogenesis phase, a scaffold of chondrocytes prefigures the skeletal elements, and the process continues until all their skeletal elements have been established. First signs of chondrogenesis in the limbs are visible once the pattern of three major regions has been established. In S. aeneus, the morphogenesis phase is evident from st33 to st35. The observations in S. aeneus are similar to those reported in lizards of genus Anolis (Sanger et al., 2012) and in the gecko P. pictus (Noro et al., 2009), but differ from those reported for C. versicolor (Mathur & Goel, 1976) and C. ocellatus (Mohammed, 1984) in which the first signs of skeletal condensation are observed early in the proximal region of limb buds. During the growth phase, the scaffold of chondrocytes increases in length, and a growth zone is established in the central region, during which ossification of the limb skeleton begins. From st36 onwards, the limbs of S. aeneus grow in length and undergo endochondral ossification, evidenced by scarce blue staining in the central region of the long bones and some phalanges. In some species, such as the geckos Taretola mauritanica and T. annularis, the ossification process is observed at early stages. Yet, in several other lizards, the first signs of osteogenesis appear in later stages of embryonic development or even after hatching (reviewed in Khannoon et al., 2024). The process of osteogenesis is evident until the later stages of embryonic development close to hatching; in S. aeneus, osteogenesis is clearly evident at st40.

Among reptiles, various limb morphological modifications have been observed. Limb reduction is a frequent modification among squamate reptiles, ranging from loss of phalanges to a complete loss of the limbs (Greer, 1991; Khannoon et al., 2024; Roscito et al., 2014; Shapiro, 2002). During embryonic development of S. aeneus, no morphological modifications of limbs were apparent. Likewise, S. aeneus manifests five digits in both forelimbs and hindlimbs with a phalangeal formula 2.3.4.5.3/2.3.4.5.4. This coincides with the ancestral pentadactyl characteristic of squamate reptiles (Greer, 1991). In this regard, an ancestral morphological pattern for limb skeletal elements has been proposed and used in studies related to limb reduction (Shapiro, 2002). Not all structures of this ancestral morphological pattern were observed in S. aeneus embryos; the pisiform element, which is associated with the ulna, was not detected in the forelimb. This observation is similar to that reported in C. versicolor, in which pisiform is detected in adult stages but not during embryonic development (Mathur & Goel, 1976). Regarding the hindlimbs, fewer elements were observed in S. aeneus than those reported in other lizards. Specifically, in Gonatodes albogularis (Leal et al., 2010), the formation of four distal tarsal (DT) elements has been reported (DT2, DT3, DT4, and DT5), while in S. aeneus, three distal tarsal elements were observed at st36 (DT2, DT3, and DT4), and two distal tarsals from st38 onwards (DT3 and DT4). The proximal tarsal element, observed in limbs of G. albogularis, is formed by the fusion of fibulare (calcaneum) and the astragalus element; previously, the astragalus is formed by the sequential fusion of three structures: the intermedium‐centrale, tibiale, and distal‐central elements (Leal et al., 2010). At st40 in S. aeneus embryos, we observed the calcified proximal tarsal element; however, the tibiale and distal central elements are not clearly defined at previous stages. Therefore, in S. aeneus embryos, we can infer that the calcified proximal tarsal element is formed by the fusion of two previous elements: the fibulare and the intermedium‐centrale. In summary, our observations show that the skeletal development of limbs in S. aeneus is similar to that observed for other reptile species (Fabrezi et al., 2007; Leal et al., 2010; Mathur & Goel, 1976; Noro et al., 2009; Sanger et al., 2012; Shapiro, 2002).

5. CONCLUSION

As discussed above, much prior work has documented embryonic development in squamate reptiles (Squamata). However, none of these works have tracked the relationship between egg size and embryonic development. In this work, we show that during the development of S. aeneus, the size of their eggs increases, and it is correlated with the embryo's stage of development. This is the first study that focuses on the growth of eggs and the developmental stage of embryos. The descriptions that we provide can be used in studies that require embryos at specific stages of development, in the analysis of biological processes or the formation and development of different organs or tissues.

AUTHOR CONTRIBUTIONS

Nivia Rocio Antonio‐Rubio: Conceptualization; investigation; writing – original draft; methodology; formal analysis; supervision. Maricela Villagrán‐SantaCruz: Writing – review and editing; funding acquisition; methodology; resources; investigation. Martha Elena Díaz‐Hernández: Writing – review and editing; methodology; investigation. Norma Moreno‐Mendoza: Funding acquisition; writing – review and editing; methodology; resources; investigation.