an enhanced developmental staging table for the …fjanzen/pdf/14jmorphol.pdfan enhanced...

An Enhanced Developmental Staging Table for thePainted Turtle, Chrysemys picta (Testudines: Emydidae)

Gerardo A. Cordero* and Fredric J. Janzen

Department of Ecology, Evolution, and Organismal Biology, Iowa State University, 251 Bessey Hall, Ames, Iowa50011

ABSTRACT Normal developmental staging tablesoften undergo expansion and enhancement in responseto advancing research paradigms and technologies. ThePainted Turtle, Chrysemys picta, has long been a pre-ferred reference taxon for comparative embryology andrecently became the first turtle species to feature asequenced genome. However, modern descriptive stud-ies on embryogenesis are lacking and an earlier devel-opmental staging table has been ignored. To addressthese problems, we re-evaluated descriptions of devel-opmental stages by studying embryos under standar-dized laboratory conditions. We created an enhancednormal developmental staging table that clarifies andvalidates previous descriptions of developmental proc-esses in this species. Moreover, we emphasized descrip-tion of turtle-specific developmental characters such asthe carapacial ridge. We demonstrated that embryogrowth rate, length of incubation period, and timing todevelopmental stages are predictable under controlledenvironmental conditions. The appearance of charac-ters associated with eye, limb, and shell anatomy wascongruent with observations made in other turtle spe-cies. To reduce experimental bias, we recommend theuse of our enhanced staging table when describingembryogenesis in the Painted Turtle. J. Morphol.275:442–455, 2014. VC 2013 Wiley Periodicals, Inc.

KEY WORDS: carapacial ridge; Chrysemys picta;embryogenesis; normal developmental staging table;shell development

INTRODUCTION

One of the first steps in biological researchrequires the observation and accurate descriptionof pattern or process. Developmental biologistsand morphologists have thus created andenhanced normal developmental staging tablesthat describe embryogenesis in a handful of repre-sentative vertebrate animals. Moreover, the crea-tion and revision of staging tables has beenmotivated by changes in technology, researchparadigms, and preference of study species (Hop-wood 2007, 2011).

Non-avian sauropsids represent a substantialportion of the amniote tree of life and are increas-ingly popular research subjects in developmentalbiology (Vickaryous and McLean, 2011; Nomuraet al., 2013). Historically, these taxa have beenoverlooked because their physiology and life his-

tory renders their embryos difficult to manipulateexperimentally (New, 1966; Billet et al., 1985).Even the application of modern developmentalassays continues to be challenging in taxa such asturtles (Gilbert, 2009). Consequently, research onthe developmental origins of morphological novel-ties, such as the turtle’s shell, has been hinderedby lack of knowledge of underlying gene regula-tory networks (Wagner and Lynch, 2010). Thesequenced genome of the Painted Turtle (Emydi-dae: Chrysemys picta; Shaffer et al., 2013) is likelyto facilitate reconstruction of gene regulatory net-works associated with shell development. Even so,modern descriptive studies on most aspects ofembryonic development are lacking for this repre-sentative model taxon. Furthermore, many impor-tant questions concerning the evolution anddevelopmental underpinnings of temporal bonearrangement of the skull (Werneburg, 2012) andsex-determining mechanisms of turtles (Janzenand Krenz, 2004; Janzen and Phillips, 2006)remain unanswered.

A lack of standardized laboratory conditions anddescriptions of embryos became problematic forcomparing and replicating studies of developmen-tal processes in the Painted Turtle (discussed inSmith, 1914; Cagle, 1954; Ewert, 1985). Earlyattempts to standardize protocols for the study ofembryos included descriptive criteria for fivestages of pectoral girdle development (Walker,1947). These stages cannot be identified reliablybecause detailed descriptions of whole embryoswere not provided and a small sample size wasused. The normal developmental staging table of

Contract grant sponsor: National Science Foundation grant; Con-tract grant number: DEB-0640932.

*Correspondence to: Gerardo Antonio Cordero; Department ofEcology, Evolution, and Organismal Biology, Iowa State, University,251 Bessey Hall, Ames, IA 50011. E-mail:[email protected]

Received 30 August 2013; Revised 8 October 2013;Accepted 13 October 2013.

Published online 3 December 2013 inWiley Online Library (wileyonlinelibrary.com).DOI 10.1002/jmor.20226

VC 2013 WILEY PERIODICALS, INC.

JOURNAL OF MORPHOLOGY 275:442–455 (2014)

Mahmoud et al. (1973) was created to addresssuch issues, but has been underutilized becauseillustrations were not highly detailed and a fluctu-ating incubation temperature (21–23�C) was used.As a result, investigators have traditionally refer-enced the staging table for the phylogeneticallydisparate Snapping Turtle (Chelydridae: Chelydraserpentina; Yntema, 1968) when describingembryogenesis in the Painted Turtle (discussed inEwert, 1985, 2008).

Analyses of developmental data generated underthe guidance of staging tables that were not mod-eled after the species of interest could be a poten-tial source of bias in comparative studies(Werneburg, 2009). For example, temporal varia-tion in developmental stages is considered a sourceof technical error in gene expression studies (Gal-lego-Romero et al., 2012). Lack of information ondevelopmental stages could also bias studies ofecological and physiological embryology in reptil-ian sauropsids (Andrews, 2004). Also, because sau-ropsid embryos are highly sensitive to theenvironment, egg incubation conditions must beaccounted for if staging criteria are to be comparedacross species (Miller, 1985). Given the imminentincrease in research centered on the Painted Tur-tle as a representative model for the study ofdevelopmental processes among amniotes (Valen-zuela, 2009), improving developmental staging cri-teria is timely and highly relevant.

The objective of this study was to enhance thedevelopmental staging table for the Painted Tur-tle. Specifically, we: (1) clarified and validated pre-vious descriptions of normal developmental stages;and (2) quantified embryonic growth rate, lengthof incubation period, and timing to respectivedevelopmental stages under highly controlled andreplicable laboratory conditions.

MATERIALS AND METHODS

During the nesting seasons of 2010–2013, 316 eggs of theWestern Painted Turtle, Chrysemys picta bellii (Gray, 1831)were collected at the Thomson Causeway Recreation Area inThomson, IL (lat. 41� 570 N; described in Schwanz et al., 2010)within the same day of oviposition. In addition, 35 eggs werecollected at the Avocet and De Fair Lake Wildlife ManagementAreas (Hyannis, NE) for scanning electron microscopy (SEM;described below). Sampling was undertaken following institu-tionally approved guidelines (protocol # 12-03-5570-J). Eggsbegan incubation under seminatural conditions (1–3 days)before transport to Iowa State University where they wereplaced in environmental chambers at a constant 27�C. Eggswere half-buried in moist (2150 kPa water potential) vermicu-lite. Weekly, the vermiculite was rehydrated and containerswere rotated to control for potential thermal gradient effects.Oxygen levels in the chambers were typically at 20–21%.

Embryos (N 5 234) were dissected from eggs and extraem-bryonic membranes using distilled water and phosphate-buffered saline before preservation. Embryos in the first weekof development were stained (in vivo) with neutral red agar tofacilitate dissection. Embryos were then fixed in 10% bufferedformalin at a mean sampling interval of 1.14 d through day 40of incubation. Some embryos of representative stages (15–21)

were also fixed in Bouin’s solution to facilitate imaging of theeye and skin. Most embryos were fixed overnight at room tem-perature and then removed for washing and dehydration usingan increasing ethanol series. Embryos from representativestages were chosen for imaging on compound (Nikon Eclipse55i) and dissecting (Nikon SMZ 745T) microscopes withmounted digital camera (Nikon DS-Vi1). Some embryos wereimaged while partially embedded in plates of 1.5% agarose inTris/Borate/EDTA. Images were edited and formatted usingprograms Preview 5.5 (Apple, Inc.), Paintbrush 2.1 (Soggy Waf-fles), and ImageJ 1.44 (NIH). Fixed embryos were staged bydirect examination on the microscope followed by review of pho-tomicrographs. Some unfixed embryos were also examined. TheMahmoud et al. (1973) staging table (MHK table hereafter) wasused to stage embryos. These descriptive criteria were re-evaluated and enhanced following generally accepted guidelinesfor the staging of reptilian embryos (Miller, 1985).

Eggs showing early signs of pipping were sampled to esti-mate total length of the embryonic incubation period. Egg pip-ping is a generally accepted index for the end of embryogenesis(Gutzke et al., 1984). Museum (ISU research collection) speci-mens of hatchling turtles were studied to describe the develop-mental stage immediately after the end of embryogenesis. Dialdigital calipers were used to record crown-rump length (CRL)and carapace length (CL) to assess the relationship betweenembryo size and developmental time (i.e., growth rate) usingregression analysis for comparison to the MHK table. Allembryo measurements were taken after fixation and dehydra-tion and choice of fixative did not appear to have an effect onsize. Statistical analyses were conducted in JMP 10.

Embryos (stages 13, 15–16) assigned to SEM imaging weresubmerged in half-strength Karnovsky’s fixative (Karnovsky,1965) and left to soak overnight at 4�C. Embryos were thendehydrated in an ethanol series and submitted to the IowaState University Bessey Microscopy and Nanoimaging Facilityfor the implementation of standard SEM methodology. Briefly,embryos were subjected to critical-point drying and sputtercoating procedures in preparation for imaging in a SEM.Images were edited and formatted as described earlier.

RESULTSRevisiting the MHK Table

Descriptions of the MHK embryonic develop-mental stages (3–22) for the Painted Turtle werevalidated and enhanced. The “hatching” stage (23)was also described. Descriptions were clarified andnew diagnostic features were provided for somestages. Embryos progressed rapidly through stages1–2; thus, observations were limited during thattime. Images of lateral or ventral aspects of wholeor parts of embryos are presented for stages 3–23(Figs. 1–9). Early formation of the apical ectoder-mal ridge (AER) of the limb bud is shown in Fig.4. Images of the carapacial ridge (CR), the turtle-specific structure involved in shell development(reviewed in Nagashima et al., 2013), are pre-sented at stages 15–16 (Fig. 6).

Stage 1 (described in MHK). The blastoporeis apparent on the dorsoposterior surface of theembryo. The chordamesodermal canal connects theblastopore to the ventral surface of the embryo.Mesoderm has begun to condense on the anteriorand posterior periphery of the embryonic disc.

Stage 2 (described in MHK). The blastoporeis U-shaped. The chordamesodermal canal

443PAINTED TURTLE DEVELOPMENTAL STAGES

Journal of Morphology

expands (anteriorly) on the ventral surface of theembryo.

Stage 3 (Fig. 1a). Somites are not visible. Theneural and head folds begin to form. The noto-chord is apparent (ventrally) and the head processbegins to flex.

Stage 4 (Fig. 1b). Three paired somites arepresent. Formation of the neural folds extendsalong the entire dorsal length of the embryo.

Stage 5 (Fig. 1c). Five paired somites arepresent. The neural folds begin to fuse anteriorly.The anterior intestinal portal has formed.

Stage 6 (Fig. 2a–b). Seven paired somites arepresent. The prosencephalon is apparent and for-mation of the optic vesicle begins. The anteriorintestinal portal is near the presumptive pericar-dial region. The neural folds have fused alongmost of the anteroposterior axis.

Stage 7 (Fig. 2c–d). Nine paired somites arepresent. The mandibular arch is apparent. Theauditory pit appears. The mesencephalon is distin-guishable. The pericardial protuberance is presentand blood islands are visible on the area vaculosa.The anterior intestinal portal has shifted caudally.

Stage 8 (Fig. 2e–f). Fourteen paired somitesare present. The stomodeum has formed and thepericardial protuberance features an S-shapedheart anlage. The lateral body folds have formedalong the entire anteroposterior axis of theembryo. The embryo is slightly oriented to the leftand has flexed dorsally. A slight tail process ispresent.

Stage 9 (Fig. 3a–b). Twenty paired somitesare present. The neuropores have closed. The firstpharyngeal slit has opened and the second andthird pharyngeal arches are visible. The maxillaand olfactory pit begin to form. The primordialheart has begun to pump blood. The optic fissureappears and formation of the lens vesicle has

begun. The embryo has now shifted its orientationto the left side (anteriorly). Early limb budsappear as condensations on the Wolffian ridge.

Stage 10 (Fig. 3c–d). Twenty five pairedsomites are present. All four pharyngeal slits arevisible and the first, second, and third ones areopen. The posterior intestinal portal has formedand the tail process is longer and curved. Limbbuds have begun to grow along the anteroposterioraxes.

Stage 11 (Figs. 3e–f, 4). Thirty paired somitesare present. All four pharyngeal slits are nowopen and the fourth pharyngeal arch is visible.Pigmentation of the retina has begun. The tailprocess is narrower distally. The allantois hasbegun to form. The limb buds are wider (antero-posteriorly) than they are long (proximodistally).The AER appears on the periphery of the limbbuds.

Stage 12 (Fig. 5a–c). Somites are difficult tocount. The fourth pharyngeal slit has closed andthe fifth has become visible. The nasolacrimalgroove and cervical sinus have formed. The uro-genital papilla appears. The limb buds are longerthan wide.

Stage 13 (Figs. 5d–f, 6a–b). External out-growth of the pharyngeal arches is highly reduced.The mandible is posterior to the eye. The retina ispigmented uniformly. The anterior intestinal por-tal has shifted to a central position on the embry-onic trunk. The limb buds assume aventroposterior orientation and are “paddle” likein form.

Stage 14 (Fig. 5g–i). The pharyngeal archesand nasolacrimal groove have disappeared. Theiris features light pigmentation. Early outgrowthof the CR is visible along the posterior flank of theembryo. The intestines have herniated into anexternal loop. The limbs feature a vaguely defined

Fig. 1. Chrysemys picta bellii, stages 3–5: embryos in ventral view (a–c; scale bars 5 1 mm).[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

444 G. A. CORDERO AND F. J. JANZEN


digital plate. The forelimb is slightly longer thanthe hindlimb.

Stage 15 (Figs. 6c–d, 7a–b). The cervicalsinus has closed. The maxilla has fused with thefrontonasal process and the external nares haveformed. The iris is entirely pigmented and theoptic fissure is no longer visible. The CR hasextended along the entire flank of the embryo andcarapacial ectoderm has begun to differentiate.The forelimb features an elbow.

Stage 16 (Figs. 6e–f, 7c–d). The caruncle hasbegun to form on the frontonasal process. The

mandible is positioned at the axial level of the pos-terior margin of the eye. Scleral papillae havebegun to form on the lower half of the retina. Con-centric growth of the CR is complete and the pri-mordial carapace now extends beyond the anteriorand posterior limb buds. The plastron has begunto form peripherally and anterior to the pericar-dial protuberance. The digital rays are visible.

Stage 17 (Fig. 7e–f). The mandible is posi-tioned at the axial level of the lens. The ectoder-mal plates (scutes) of the carapace are delineatedand the plastron extends over and across a much

Fig. 2. Chrysemys picta bellii, stages 6–8: left column, embryos in ventral view (a, c, and e; scalebars 5 1 mm). Right column, detail view of head and pharyngeal regions (b, d, and f; scale bars5 100lm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



reduced pericardial protuberance. The AER isreduced and serration of the digital plate hasbegun. Sparse pigmentation is visible at the baseof the limbs.

Stage 18 (Fig. 8a–b). The mandible is posi-tioned beyond the axial level of the anterior mar-gin of the lens. Scleral papillae cover the entireretina and the lower eyelid is present. The plas-tron has completed its growth along the entireanteroposterior axis of the embryo. The carapacialscutes are clearly differentiated. Interdigital webshave begun to differentiate on the digital plate.

Stage 19 (Fig. 8c–d). The anterior margin ofthe mandible is positioned between the eye andfrontonasal process. The lower eyelid has begun togrow over the sclera and the upper eyelid is pres-

ent. Pigmentation of the carapacial scutes hasbegun. The digits extend beyond the interveningwebbing. Pigmentation of the limbs has increased.

Stage 20 (Fig. 8e–f). The rhampothecae aredifferentiated. Scleral papillae are no longer visi-ble. Cutaneous papillae have begun to grow on theneck. Plastral scutes are delineated and marginalcarapacial scutes feature a pigmented line. Thelimbs feature ectodermal scales and presumptiveclaws are lightly pigmented. The cloaca is fullydifferentiated.

Stage 21 (Fig. 9a–b). The mandible is at itsocclusion point with the maxilla. The upper eyelidhas begun to grow over the sclera and the nictitat-ing membrane has formed. Cervical constriction isreduced and a skin fold has formed on the dorsal

Fig. 3. Chrysemys picta bellii, stages 9–11: left column, embryos in lateral view (a, c, and e; scalebars 5 1 mm). Right column, detail view of head and pharyngeal regions (b, d, and f; scale bars5 1mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



neck. A lightly pigmented line demarcates the dor-sal midline of the carapace. Both limbs featurelightly pigmented stripes. Claws feature skin foldsproximally.

Stage 22 (Fig. 9c–d). Both eyelids extendtheir growth over the iris. The head features pig-mented stripes. The carapacial keel has formed.The plastron is pigmented, particularly around theumbilicus. The intestinal loop is withdrawn. Thelimbs and tail are thoroughly pigmented and uni-formly covered by scales interspersed by skinfolds.

Stage 23 (Fig. 9e–f). The pupil has darkened.Pigmentation (yellow and dark olive) of the cara-pace, plastron, head, tail, and limbs has increased.The number of scales on the head, limbs, and tailhas increased. The turtle has hatched from itsegg.

Embryo size, Growth Rate, and IncubationPeriod

Variation in embryo size was recorded beginningat stage 11 (CRL; Table 1). Beginning at stage 15,embryo size was estimated using CL (Fig. 10;Table 1). Variation in CL was strongly predictedby incubation period (Fig. 11; polynomial fit,R2 5 0.97, N 5 131, F2,128 5 2490, P<0.0001).Means for embryo size at 27�C (this study;R2 5 0.99, N 5 8, F2,5 5 446, P< 0.0001) and 21–23�C (Mahmoud et al. 1973; R2 5 0.95, N 5 8,

F2,5 5 51.3, P<0.001) were strongly predicted byincubation period in regression models fitted withpolynomial curves (Fig. 12). Length of incubationperiod was 53.5 d at 27�C.

DISCUSSIONEnhancement and Validation of the MHKTable

The descriptive criteria of the MHK table, whichwere roughly modeled after Yntema’s (1968) stag-ing table for the Snapping Turtle, met the basicpremise for the designation of developmentalstages in vertebrate embryos (Hamburger andHamilton, 1951) stages were clustered as closelypossible and could be unequivocally identified byat least one external character. In addition, ourenhanced criteria satisfy guidelines for the estab-lishment of developmental stages in reptilian sau-ropsids (Miller, 1985) because we carefullycontrolled for environmental variables (gas, mois-ture, substrate, and temperature) during incuba-tion. Although our observations were generallycongruent with the MHK table, we did note someimportant differences. We observed the early for-mation of a tail process in stage 8 embryos ratherthan at stage 7. Also, the AER, which is first men-tioned in stage 14 of the MHK table, became appa-rent on both limb buds at stage 11. Moreover, weobserved earlier onset of digital plate serration(stage 17) than described in the MHK table (stage

Fig. 4. Chrysemys picta bellii, stages 11: dorsal view of right forelimb bud (scale bar 5 100 lm)and detail view of the apical AER (scale bar 5 100 lm). [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]



18). The MHK table does not indicate the onset ofclaw differentiation, although completion of clawdevelopment is described in stage 22. We describedthe onset of claw differentiation and its increasedpigmentation at stages 20 and 21, respectively.Because completion of mandibular developmentwas not indicated in the MHK table (discussed inGreenbaum and Carr, 2002), we described the ter-minal differentiation of that structure at stages20–21.

By carefully tracing the development of otherstructures, such as the eye, we noted stage-specificdiagnostic features that were not reported in theoriginal MHK table. For example, we describedthe early onset of scleral papillae and their growtharound the retina (stages 16–20), whereas thischaracter is first mentioned at stage 19 of theMHK table. We also made new observations on

the appearance of a nictitating eye membrane dur-ing stage 21 and change in color of the pupil atstage 23. Overall, the original work of Mahmoudet al. (1973) and our enhanced description of theirstaging table should provide solid guidance for thestudy of embryogenesis in the Painted Turtle.

Accounting for Incubation Environment

Growth rates of vertebrate embryos can behighly variable within and among species anddepend greatly on incubation temperature (Reiss,1989). Investigators have thus relied on stagingtables to reduce experimental error due to thisvariation (Hall and Miyake, 1995). We incubatedembryos of the Painted Turtle under controlledlaboratory conditions, which included a constant

Fig. 5. Chrysemys picta bellii, stages 12–14: left column, embryos in lateral view (a, d, and g; scale bars 5 1 mm). Middle column,detail (dorsal) view of right forelimb buds (b, e, and h; scale bars 5 100 lm). Right column, detail view of head and pharyngeal region(c, f, and i; scale bars 5 1 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



temperature (27�C) within the range of those usedby most investigators (Fig. 13) and typical of nesttemperatures in our Illinois population (Weisrockand Janzen, 1999). Temperature strongly influen-ces embryonic growth rate and duration of incuba-tion period in this ectothermic sauropsid (Janzenand Morjan, 2002). Substrate moisture and oxygenavailability can also affect growth rate and incuba-tion period (reviewed in Packard and Packard,1988). The methodology used to establish stages ofthe MHK table did not carefully control for thesevariables, as in many classical works on reptilianembryology (Miller, 1985). Such neglect of theincubation environment has likely confoundedattempts to compare developmental chronology in

turtles (e.g., Renous et al., 1989; Tokita and Kura-tani, 2001).

We found that incubation period, and thusduration and timing to developmental stages,might be replicable when controlling for hydricand thermal environment. Our estimate of incu-bation period was the same (53.5 d) as that of astudy that sampled eggs from a similar latitude(42� 520 N) under similar laboratory conditions(53.5 d at constant 27�C and 2150 kPa; Gutzkeet al., 1987). Developmental variation might notbe explained solely by environment, as weobserved embryo size variation within stages.Even so, we more effectively characterized embry-onic growth rate and with larger sample sizes

Fig. 6. Chrysemys picta bellii, scanning electron micrographs of stages 13, 15–16: left column, detail views of the posterolateral(left side) region of embryos where the CR forms (a, c, and e; scale bar 5 1 mm). Right column, dorsal view of right forelimb buds fea-turing the apical AER (b, d, and f; scale bar 5 1 mm). Arrows (bottom, middle column) indicate planes of orientation (A 5 anterior,P 5 posterior, D 5 dorsal, V 5 ventral).



than Mahmoud et al. (1973). Future studiesshould carefully address incubation environmentor adjust experimental designs accordingly. Also,we recommend increased awareness of embryo-genesis in the Painted Turtle and other reptiliansauropsids, as it could inform current knowledgeon phenotypes that are strongly affected by theincubation environment (e.g., temperature-dependent sex determination; discussed inAndrews, 2004).

Comparisons to Other Turtle Species

Our observations of embryogenesis in the PaintedTurtle not only enhance its developmental staging

table, but also disprove assumptions made in previ-ous comparative analyses. For example, followingdescriptions from the MHK table, Tokita and Kura-tani (2001) speculated that embryogenesis in thePainted Turtle was “unique,” presumably due todelayed development of the carapace, eye, limb, andplastron. Although those authors had only observedembryogenesis in the Chinese Softshell Turtle (Trio-nychidae: Pelodiscus sinensis), they claimed thatdevelopment in the Painted Turtle differed in com-parison to that species, the Snapping Turtle, andmarine turtles. We clarified that the timing of dif-ferentiation for the carapace, eye, limb, and plas-tron is not delayed in the Painted Turtle.

Fig. 7. Chrysemys picta bellii, stages 15–17: left column, embryos in lateral view (a, c, and e;scale bars 5 1 mm). Right column, detail view (dorsal) of right forelimbs (b, d, and f; scale bars5 100lm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



We confirmed that formation of the AER pre-cedes digital plate differentiation, as demonstratedin most turtle species. This structure was alreadyapparent in embryos of the Painted Turtle at thesame developmental stage (12) as in the ChineseSoftshell Turtle (Tokita and Kuratani, 2001) andSnapping Turtle (Yntema, 1968). Similarly, theappearance and maturation of the CR did not dif-fer in the Painted Turtle. Our direct examinationof embryos of the Snapping Turtle and Spiny Soft-shell Turtle (Apalone spinifera), a close relative ofthe Chinese Softshell Turtle, supports that pattern(Cordero, 2012; unpublished data). We observedsome aspects of eye, limb, and shell differentiation

earlier than in the MHK table, which was consistentwith staging tables for the closely related emydidRed-eared Slider (Trachemys scripta, Greenbaum,2002), as well as the Chinese Pond Turtle (Chinemysreevesii, Tan et al., 2001) and the Japanese PondTurtle (Mauremys japonica, Okada et al., 2011) ofthe sister family Geoemydidae. We demonstratedthat important developmental processes are notdelayed in the Painted Turtle relative to other stud-ied turtles.

Overall, timing of differentiation of the basic tur-tle body plan and externally positioned sensoryorgans appear to be highly conserved across deeplydivergent turtle lineages. However, we are cautious

Fig. 8. Chrysemys picta bellii, stages 18–20: left column, embryos in lateral view (a, c, and e;scale bar 5 1 mm). Right column, detail view (dorsal) of right forelimbs (b, d, and f; scale bars 5 1mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



about these generalizations, as development hasonly been studied in a handful of taxa. Develop-mental staging tables or systems exist for represen-tatives of 10 of 14 recognized turtle families (Danniet al., 1990; Okada et al., 2011; reviewed in Tokitaand Kuratani, 2001; Greenbaum and Carr, 2002;Werneburg et al., 2009). Based on this work, wededuced that clade-specific phenotypic differencesare generally established during the middle one-third of embryogenesis. We observed that embryosof the Painted Turtle could be distinguished, basedon relative body size, craniofacial morphology, pig-mentation, and tail length, from those of Chelydri-dae and Kinosternidae as early as stage 17. Incontrast, they were indistinguishable from those ofMap Turtles (Emydidae: Graptemys) until stage 21(Cordero, 2011; unpublished data). Because differ-

ences in phylogenetically disparate species becomeapparent earlier in embryogenesis (e.g., SnappingTurtle vs. Painted Turtle), we recommend the useof staging tables designed specifically for the spe-cies of interest or a close relative.

Embryogenesis in the closely related PaintedTurtle and Red-eared Slider is highly similar,except for differences in the latest stages (seeGreenbaum, 2002). The Red-eared Slider has alonger incubation period and reaches larger hatch-ling and adult body sizes. Theory predicts thatspecies with larger adult body size should havelonger developmental incubation periods (Reiss,1989), which could explain additional stages ofdevelopment (24–26) described in the Red-earedSlider. These stages are characterized by minorspecies-specific differences in pigmentation

Fig. 9. Chrysemys picta bellii, stages 21–23: left column, embryos in lateral view (a, c, and e;scale bar 5 2 mm). Right column, detail view (dorsal) of right forelimbs (b, d, and f; scale bars 5 1mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



patterns and ectodermal morphology. For example,a translucent sheath on the ventral claw of theRed-eared Slider was described, although we didnot observe it in embryos of the Painted Turtle.This character was also observed in the late devel-opmental stages (24–26) of the Japanese PondTurtle (Okada et al., 2011) and Snapping Turtle(Yntema, 1968), both of which are larger and havelonger incubation periods than the Painted Turtle.

The staging table for the Red-eared Slider wasdesigned to mirror the descriptive criteria ofYntema (1968). For example, both tables describethe appearance of scattered pigment cells on stage15 limb buds. Yet this early onset of limb pigmen-tation in the Red-eared Slider would seem to be

unusual, as it is not apparent until stage 17 in thePainted Turtle, although we have observed pig-mentation as early as stage 16 in the SnappingTurtle. Efforts to adhere to the criteria of Yntema(1968) may have biased descriptions in the stagingtable of the Red-eared Slider. Our staging table isbetter suited to describe embryogenesis in emydidturtles before species-specific differences are estab-lished late in development. We acknowledge thatsome aspects of developmental staging tables areinherently subjective and encourage the combineduse of alternative approaches, such as the Stand-ard Event System for vertebrate embryology

TABLE 1. Chrysemys picta bellii, Embryo size, developmental chronology, and defining characters of developmental stages

Stage CRLa (mm 6 SEb) CLc (mm 6 SEb) N Day(s) Week(s) Defining characterd

3 4 3 1 Notochord4 3 4 1 Somites5 6 5 1 Anterior intestinal portal6 7 6 1 Optic vesicles7 9 7 1 Mandibular arch8 8 7–8 1–2 Stomodeum9 6 7–9 2 First pharyngeal slit is open10 5 10 2 Posterior intestinal portal11 5.57 (0.30) 5 10–12 2 Limb buds with apical ectodermal ridge12 6.49 (0.23) 7 10–15 2–3 Urogenital papilla13 7.01 (0.14) 6 11–16 2–3 Retina is uniformly pigmented14 7.30 (0.30) 6 14–17 3 Carapacial ridge15 6.06 (0.42) 12 16–18 3 External nares16 6.53 (0.13) 11 17–21 3–4 Caruncle17 7.30 (0.12) 14 18–23 3–4 Serration of digital plate18 8.67 (0.20) 18 22–27 4 Interdigital webs19 10.2 (0.37) 17 24–31 4–5 Carapace pigmentation20 12.2 (0.32) 14 26–33 4–5 Claws21 15.6 (0.26) 20 27–36 4–6 Pigmented stripes on limbs22e 18.5 (0.18) 27 37–53 6–8 Plastron pigmentation

aCRL 5 Crown-rump length (mm) measurements were used to estimate embryo size during stages 11–14.b6 SE 5 Standard error.cCL 5 Carapace length (mm) was used as an index of embryo size during stages 15–22.dA character that first appears or undergoes change during the listed stage of development.eSize measurements were taken when embryos first reached stage 22 (�37–38 d). Embryos (N 5 5) in pipped eggs (i.e., immedi-ately before hatching; �53.5 d) were>22 mm in carapace length.

Fig. 10. Chrysemys picta bellii, variation in size (estimated byCL) of embryos at stages 15–22. * 5 Measurements were takenwhen embryos first reached that stage (�37–38 d).

Fig. 11. Chrysemys picta bellii, CL as a function of incubationperiod (at 27�C).



(Werneburg, 2009). In either case, diligent exami-nation of embryonic anatomy is necessary toreduce bias in comparative studies. Furthermore,staging tables are based primarily on externalcharacters; less is known about developmental dif-ferences that define diversity in the internal anat-omy of turtles.

In summary, we recommend that investigatorsof the Painted Turtle preferentially use ourenhanced developmental staging table over thosedesigned for the Snapping Turtle (Yntema, 1968)and Red-eared Slider (Greenbaum, 2002) for the

following reasons: (1) estimates on the chronologyof developmental stages could be affected by longerincubation periods in these species; (2) Species-specific developmental differences, both early(stage 17, Snapping Turtle) and late (stages 24–26,Red-eared Slider) could bias descriptions and,thus, staging of embryos.

ACKNOWLEDGMENTS

The authors thank members of the Janzen labwho assisted with sample collection in Illinois andAnthony Beringer and Joyce Lok, who assistedwith sample collection in Nebraska. Sampling wasconducted with corresponding scientific collectionpermits for Illinois (NH13.0073) and Nebraska(310). The thank Christan Halverson for labora-tory assistance and Tracey Pepper (ISU BesseyMicroscopy and Nanoimaging Facility) for helpwith SEM procedures. Authors thank anonymousreviewers whose comments helped to improve anearlier version of this manuscript. GAC was sup-ported by the ISU-GMAP fellowship.

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