JOURNALOFMORPHOLOGY254:272–291(2002) StagingCriteriaforEmbryosoftheSpinySoftshell Turtle,Apalonespinifera(Testudines:Trionychidae) EliGreenbaumandJohnL.Carr* DepartmentofBiology,TheUniversityofLouisianaatMonroe,Monroe,Louisiana71209-0520 ABSTRACTPreviousworkdescribingtheembryonic schemesubsequentlyfollowedbyRenousetal.(1989) stagesofturtledevelopmenthasnotincludedmembersof forD.coriaceaandBillettetal.(1992)forC.caretta. thehighlyderivedtrionychidturtles.Stagingcriteriaare TheseminalworkofEwert(1985)onturtleem- describedforthespinysoftshellturtle(Apalonespinifera) bryologyemployedthestagesofYntema(1968)for tofacilitatecomparisonsbetweenphylogeneticallydistant taxaofturtles.EmbryonicdevelopmentinA.spiniferais descriptiveandcomparativepurposes,including placedinthecontextofthewidelyusedsequenceof considerableefforttointerprettheolder,classic Yntemastages.Novelfeaturesareincludedinthedescrip- studiesofdescriptiveembryologyintermsof tionsofstagingcriteriaforStages13–26.Comparisonsof Yntema’sstages.Ewert(1985)extendedtheuseof thedevelopmentofspecificfeaturesaremadebetweenA. theYntemacriteriatoincludeparticularstageswith spiniferaandothertaxaofturtles.Dataonthedurationof highlyvisiblecharacteristicsthatcouldbediscerned developmentalstagesatdifferenttemperaturesandem- bycandlingofeggs,thusallowingapproximateag- bryodimensionssupporttheconclusionthatmorphology- ingofembryoswithouttheirsacrifice.Thisapproach basedstagingcriteriaaresuperiortodevelopmentalrate temperaturecoefficients.J.Morphol.254:272–291,2002. waselaboratelydevelopedforCarettochelysin- ©2002Wiley-Liss,Inc. sculptainconcertwithasummarynormalseriesin termsofYntemastages(Beggsetal.,2000).Many KEYWORDS:Testudines;Trionychidae;Apalonespini- studieshaveroutinelyagedturtleembryosusing fera;embryology Yntemastages,suchasvariousreproductivestudies (e.g.,PieauandDorizzi,1981;RaynaudandPieau, 1985;Wibbelsetal.,1991).Inaddition,Guyotetal. Priortoformaldescriptionandstandardizationof (1994)describedanormalseriesusingYntema- theembryonicstagesofdevelopmentinturtles,re- equivalentstagesforthetortoiseTestudohermanni. searchersusedmeasurementrangestodescribeap- Inordertofacilitatecomparisonofstructuresor proximateagesofembryos(e.g.,Risley,1933). eventsinthedevelopmentofanyrelatedgroupof Yntema(1968)introducedtheuseofanormative organisms,astandardsetofdevelopmentalstagesis seriesofdevelopmentalstagesforturtleembryos essential.AlthoughYntema’s(1968)stagingseriesis withhisdescriptionof27stages(0–26)inthedevel- widelyusedforturtles,trionychidslacksomekeymor- opmentofChelydraserpentina.Hisstageswere phologicalfeaturesowingtotheirhighlydivergent basedontimedperiodsofdevelopmentatagiven morphology.Forexample,carapacialscutesareabsent constanttemperature.Foreachstageheprovideda andthereisageneralreductionofkeratinizedinteg- detaileddescriptionoftheexternalmorphologyand umentarystructures,includingthepresenceofonly suggestedthatsomeofthecharacteristicswould threedigitswithclawsperlimb.Someprominentat- serveasdifferentialcriteriaforstagerecognition. tributesoftrionychidshaveneverbeenusedinan SincethepublicationofYntema’s(1968)staging embryonicstagingseries(e.g.,lipsalongtheupperand criteria,severalothernormalserieshavebeende- scribedforvariousturtles;however,thisprolifera- tionhastendedtocomplicateratherthanfacilitate makingtaxonomiccomparisons.Forexample,in Contractgrantsponsors:SigmaXi,theScientificResearchSociety, theirstudyoftheemydidturtleChrysemyspicta, theChicagoHerpetologicalSociety(EG). Mahmoudetal.(1973)described23,ratherthan27, CurrentaddressforE.Greenbaum:DivisionofHerpetology,Natu- developmentalstages.Crastz(1982)described31 ralHistoryMuseum&BiodiversityResearchCenter,TheUniversity developmentalstagesinthecheloniidLepidochelys ofKansas,1345JayhawkBoulevard,Lawrence,Kansas66045-7561. olivaceabasedonmorphologyandmeasurements ofembryos.Miller(1985)departedfrompriorau- *Correspondenceto:JohnL.Carr,DepartmentofBiology,theUni- thorsbyincludingpreovipositionalstagesamong versityofLouisianaatMonroe,Monroe,LA71209-0520. his31stagesofdevelopmentinseveraltaxaof E-mail:[email protected] embryonicseaturtles(Cheloniamydas,Natator Publishedonline00Month2002in depressa,Carettacaretta,Eretmochelysimbricata, WileyInterScience(www.interscience.wiley.com) Lepidochelysolivacea,andDermochelyscoriacea),a DOI:10.1002/jmor.10036 ©2002WILEY-LISS,INC. APALONE SPINIFERA EMBRYOLOGY 273 lower jaws), but there are few published studies in- cies, the ranges of which meet in the area. The specimens from West volving embryonic trionychids. Ling et al. (1985) did Feliciana Parish are consistent with populations that Webb consid- ered intermediate in characterization between A. s. spinifera and A. not stage embryos of the Asian trionychid Pelodiscus s. hartwegi, with some possible influence of A. s. aspera. Ouachita sinensis in their study of epiplexus cells; embryonic Parish specimens apparently are intergrades between A. s. spinifera age (in days) was used in place of a staging scheme. and A. s. hartwegi and those from Concordia Turtle Farm exhibited Cherepanov (1995) referred to ranges of Yntema somewhat more A. s. spinifera influence. We expect that the mor- stages (e.g., 18–21) in a study of shell development in phological characteristics mentioned in this work are valid for all subspecies of A. spinifera, with the possible exception of carapace the same species. Webb et al. (1986) attempted to pigmentation in A. s. pallida, A. s. guadalupensis, and A. s. emoryi. stage embryos of Carettochelys insculpta, the sister Embryos incubated in 1996 (96A, B) were sacrificed starting on 26 group of the trionychids (Shaffer et al., 1997), with June 1996 by severing the spinal cord. Embryos from 1997 (97A, B) metric data (e.g., head measurements) that are only were sacrificed by immersion in a 0.2% solution of MS-222 if they were less than 1 cm in total length, or by intracardiac injection of vaguely comparable to Yntema stages. Beggs et al. sodium pentobarbital if they had a total length exceeding 1 cm (2000) devised a Carettochelys staging series meant to (AVMA Panel on Euthanasia, 1993). Embryos from 1996 (96A, B) coincide with that of Yntema (1968) and they de- were preserved in 10% formalin for 12 months before transfer to scribed characters taking into account its unique mor- Bouin’s solution for at least 2 weeks, then stored in 50% isopropyl phology, some of which are shared with trionychids, alcohol (Presnell and Schreibman, 1997). Embryos from 1997 (97A, B) were preserved in Bouin’s solution for at least 2 weeks before e.g., the lack of carapacial scutes. transfer to 50% isopropyl alcohol for long-term storage. A total of 32 Herein, we describe an objective scheme of mor- embryos was harvested from the 1996 clutches and 101 embryos phological stages for the trionychid Apalone spini- were collected from the 1997 (97A, B) eggs. Several turtles were fera meant to correspond with the Yntema series. allowed to hatch from each clutch of eggs (to be used in later stud- ies). The remaining eggs were either infertile or the embryos died in We assume that the earliest developmental stages situ due to unknown causes. are common to all turtles (based on fundamental Embryos from Stages 11–15 were definitively staged according to features found in all turtle taxa); therefore, we em- Yntema’s (1968) criteria. Embryos beyond these stages were classi- phasize the second half of embryonic development, fied with morphological characters including backlit claw structure, during which time the distinctive trionychid fea- nasal structures, urogenital papilla development, labial develop- ment, and nictitating membrane appearance. Claw structure is de- tures are formed. Another objective is to compare scribed from the second digit in dorsal view, as seen by shining a the several published normal series for turtles and bright light through the translucent digit. The duration of stage for development of selected features across taxa. each temperature was calculated by plotting each staged embryo on a timeline (in days) for each clutch of eggs separately for each temperature. We divided the elapsed time equally among the in- MATERIALS AND METHODS terim stages and assigned that value to each embryo for those stages involved. The average duration of each stage was calculated by A total of 306 eggs of Apalone spinifera was collected for the obtaining the mean of all embryo-specific stage durations. Following purpose of this study. Forty-four eggs (collected from Big Bayou stage classification, embryos were photographed and placed in 50% Sara and Little Bayou Sara, West Feliciana Parish, Louisiana, in isopropyl alcohol. The kidney/gonad complex was extracted for use the second half of May 1996, hereafter denoted 96A) and 46 eggs in a companion study of gonadogenesis (Greenbaum and Carr, (collected from nests or gravid females from Ouachita Parish, 2001). Head and carapace lengths of the embryos were measured hereafter denoted 96B) were maintained at a steady temperature with dial calipers to the nearest 0.05 mm. Carapace length mea- of 24.5°C until 10 June 1996, when all eggs were moved to an surements before Stage 16 were not taken because the carapace is incubator. Thirty-six eggs were obtained from nests or gravid not fully formed until this stage. females from Black Bayou Lake in Ouachita Parish, Louisiana, in May and June 1997 (hereafter denoted 97A) and an additional 180 eggs were obtained on 4 July 1997 from Concordia Turtle RESULTS Farm in Wildsville, Louisiana (hereafter denoted 97B). All eggs were individually numbered and placed in plastic boxes