Ontogenetic sequence reconstruction and sequence polymorphism in extinct taxa: an example using early tetrapods (Tetrapoda: Lepospondyli)

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Ontogenetic sequence reconstruction and sequencepolymorphism in extinct taxa: an example using early tetrapods(Tetrapoda: Lepospondyli)Author(s): Jennifer C. OloriSource: Paleobiology, 39(3):400-428. 2013.Published By: The Paleontological SocietyDOI: http://dx.doi.org/10.1666/12031URL: http://www.bioone.org/doi/full/10.1666/12031

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Ontogenetic sequence reconstruction and sequence polymorphism in extincttaxa: an example using early tetrapods (Tetrapoda: Lepospondyli)

Jennifer C. Olori

Abstract.— Ontogenetic sequence reconstruction is challenging particularly for extinct taxa because ofwhen, where, and how fossils preserve. Different methods of reconstruction exist, but the effects ofpreservational bias, the applicability of size-independent methods, and the prevalence of sequencepolymorphism (intraspecific variation) remain unexplored for paleontological data. Here I comparefive different methods of ontogenetic sequence reconstruction and their effects on the detection ofsequence polymorphism, using a large collection of the extinct vertebrates Microbrachis pelikani andHyloplesion longicostatum. The postcranial ossification sequences presented here for those taxa are thefirst examples known for extinct lepospondyls. Sequences were reconstructed according to skull length,trunk length, increasing number of ontogenetic events, majority-rule consensus, and OntogeneticSequence Analysis (OSA). Results generally were in agreement, demonstrating that paleontologicaldata may be used to robustly reconstruct developmental patterns. When reconstructing sequencesbased on fossils, size-based methods and OSA are more objective and less dependent on preservationalbias than other techniques. Apart from the other methods, OSA also allows for statistical analysis ofobserved and predicted polymorphism. However, OSA requires a large sample size to yield meaningfulresults, and size-based methods are justified in paleontological studies when sample size is limited bypoor preservation. Different methods of reconstruction detected different patterns of sequencepolymorphism, although across all methods the magnitude of sequence variation for M. pelikani and H.longicostatum (1.3�3.4%) was within the lower range of values reported for extant vertebrates.Compared with other extinct and extant tetrapods, all sequence reconstruction methods consistentlyshowed that M. pelikani and H. longicostatum exhibit advanced ossification of the pubis and delayedossification of the scapula. However, the postcranial ossification sequences of these two taxa largely arecongruent with those of other tetrapods, suggesting an underlying conservative ancestral pattern thatevolved early in tetrapod history.

Jennifer C. Olori.* Department of Geological Sciences, Jackson School of Geosciences, University of Texas atAustin, Austin, Texas 78712, U.S.A. E-mail: [email protected]. *Present address: Department ofBiological Sciences, State University of New York at Oswego, Oswego, New York 13126, U.S.A.

Accepted: 7 March 2013Published online: 3 May 2013Supplemental materials deposited at Dryad: doi 10.5061/dryad.4gv88

Introduction

The ontogenies of organisms, particularly

ontogenetic sequences, have become a majorsource of comparative information for under-

standing the relationships and evolutionary

history of extinct taxa (e.g., Hughes et al. 2006;Schoch 2006; Anderson 2007; Frobisch et al.

2007; Hunt and Yasuhara 2010). However,reconstruction of ontogenetic sequences based

on fossils is difficult and problematic. In

studies of extant taxa a growth series can beobtained through a longitudinal study of the

same, living individuals tracked throughouttheir lives, or by utilizing specimens con-

trolled for age or stage. The first method isimpossible for studies relying on fossils and

the second highly unlikely because of preser-

vational bias. Thus, in most paleontologicalresearch a growth series is reconstructed fromthe compilation of available specimens, andthe ontogenetic sequence is reconstructedaccording to specimen size. Despite therecognition that size frequently is not corre-lated with maturity within a species (Hallidayand Verrell 1988; Jones 1988; Brandt 1991; Yeh2002), the use of size as a proxy for age ormaturity remains the most common methodof obtaining developmental information fromextinct taxa, and few size-independent tech-niques have been explored in paleontologicalstudies (but see Brochu 1996; Colbert 1999;Colbert and Rowe 2008). Poor preservation isan additional compounding problem specificto developmental studies that rely on fossils.Fossils frequently are incomplete or disarticu-

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Paleobiology, 39(3), 2013, pp. 400–428DOI: 10.1666/12031

lated, adding another obstacle to the alreadychallenging task of reconstructing ontogeneticsequences of extinct organisms.

Moreover, although it is acknowledged thatintraspecific developmental variation exists,and research on populations of extant taxademonstrates that variation can have a pow-erful and meaningful presence (Sheil andGreenbaum 2005; Colbert and Rowe 2008;Bever 2009), few studies have explored thedetection and magnitude of ontogenetic se-quence polymorphism in collections of fossils(but see Hunt and Yasuhara 2010). Addition-ally, we know little about how the magnitudeof variation detected for extinct taxa compareswith that in extant taxa, and how sample sizerelates to both apparent polymorphism andsequence reconstruction.

The primary purpose of this study was toevaluate different methods that have beenproposed for the reconstruction of ontogeneticsequences for extinct taxa, and to documentand compare potential sequence polymor-phisms identified under the different methods.

As a case study that is applicable for trilobitesto Tyrannosaurus rex, a large assemblage ofMicrobrachis pelikani and Hyloplesion longicos-tatum at different sizes and maturity levelspresented a rare opportunity to evaluatemultiple methods of sequence reconstruction,assess the repeatability (robustness) of se-quence reconstructions in extinct taxa, andquantify the magnitude of intraspecific varia-tion. Historically, M. pelikani and H. long-icostatum were considered to be members of‘‘Microsauria,’’ one of a number of groups ofdiminutive early tetrapods (e.g., Aistopoda,Nectridea, Lysorophia) included in the cladeLepospondyli (Carroll and Gaskill 1978; Car-roll 2000). However, ‘‘microsaurs’’ now arerecognized to form a paraphyletic grade withrespect to other members of the Lepospondyli(Fig. 1). For a long time, even the monophylyof lepospondyls was questioned, although allrecent large-scale phylogenetic analyses con-sistently indicate a monophyletic Lepospon-dyli that is more closely related to amniotesthan to temnospondyls (Ruta and Coates 2007;

FIGURE 1. Simplified phylogeny of higher level relationships of early tetrapods based on Maddin et al. (2012) and Rutaand Coates (2007). Note that relationships within Lepospondyli still are unstable and the 30þ species of ‘‘microsaurs’’ arerepresented as two composite clades (triangles).

ONTOGENETIC SEQUENCE RECONSTRUCTION 401

Anderson et al. 2008; Sigurdson and Green2011; Maddin et al. 2012).

Almost all published patterns of the growthand development of Paleozoic tetrapods comefrom studies on temnospondyls (e.g., Bystrowand Efremov 1940; Milner 1982; Schoch 1992,2002, 2003, 2004; Witzmann and Pfretzschner2003; Witzmann 2005, 2006; Witzmann andSchoch 2006; Frobisch et al. 2007; Frobisch2008), one of the major clades linked to theancestry of extant amphibians (Ruta et al.2003; Ruta and Coates 2007; Anderson et al.2008). However, lepospondyls also werelinked to the origin of amphibians (Vallinand Laurin 2004; Anderson et al. 2008) anduntil now, only a single study addressedossification sequences in lepospondyls (Phle-gethontia [Anderson 2002]). The paucity ofontogenetic data for non-temnospondyl earlytetrapods has the potential to bias inferencesabout the evolution of development andphylogenetic relationships within Tetrapoda.Here I provide the first description of thepostcranial ossification sequence of any lep-ospondyl; specifically I reconstruct the ossifi-cation sequence of the limbs and girdles for M.pelikani and H. longicostatum, and provideadditional information on the first develop-mental appearance of haemal arches, scleralossicles, and branchial plates in these species.Sequences reconstructed for these taxa also arecompared with one another, as well as with arepresentative sample of extinct and extanttetrapod sequences obtained from the litera-ture.

Material and Methods

All specimens of M. pelikani and H. long-icostatum included in this study were collectedfrom a single locality and deposit, the upperCarboniferous ‘‘Gaskohle’’ of the HumboldtMine, Nyrany, Czech Republic (Fric 1876). Thefossil-bearing layer is contained within a 30-cm-thick package of finely laminated coals,shales, and mudstones (‘‘Plattelkohle’’) thatdoes not represent a single depositional event,but it is confined to a period of gradualdeposition not thought to exceed 700 years(Milner 1980). Hundreds of small tetrapodfossils were discovered within 1 cm of adistinct 2–5 cm band of mudstone (Milner

1980). From studies of stratified lakes andswamps present in the same area today,Skocek (1968) calculated that 4.4�10.8 cm oflithified mudstone represents 100 years ofsedimentation, suggesting that the tetrapodlayer was formed over 100 years or less(Milner 1980). As such, the Nyrany depositcaptures many life stages of M. pelikani and H.longicostatum, thus having an advantage oversingle-event deposits that may trap only onestage, such as breeding adults or small larvae.Because of the small period of time represent-ed, the samples can be regarded as a record ofsingle populations amassed over short geo-logic time, an interpretation supported by thepresence of autapomorphies in individuals ofeach taxon.

The deposit containing the fossils formedunder anoxic conditions, preventing benthicorganisms from disturbing burial and decom-position. As a result, the majority of specimensare preserved in articulation or with skeletalelements in close association. Because the coallayers split apart easily, specimens often arepreserved as part and counterpart, each halfretaining various proportions of bone andimpressions. Previous researchers often etchedaway all bone to produce detailed latex casts(Baird 1951), which sometimes is the onlypractical method to study the specimens. Evenwhen bone is removed the specimens still arepreserved as finely detailed impressions with-in the coal. Frequently the detail is exception-al, capturing bone sculpture, dermal scaleorganization and ornamentation, lateral linecanals, sclerotic plates, small foramina, andthe outline of tiny terminal phalanges. In thosecases it is clear when bones are absent becausethey were unossified rather than disarticulat-ed and destroyed. In many specimens, forexample, the long bones, metacarpals, andphalanges are preserved in association but adistinct gap occurs where the epipodials hadnot yet ossified. Long-term preservation,however, was an important factor; where bonewas left in place, subsequent degradation bypyrite or gypsum crystallization resulted in alarge amount of missing data in manyspecimens.

Growth series based on 77 individuals of M.pelikani and 12 individuals of H. longicostatum

402 JENNIFER C. OLORI

were assessed for the presence of ossifiedpostcranial elements. In order to reconstructontogenetic sequences, I inferred relativematurity in five different ways: according toskull length, trunk length, a combination ofsize and increasing number of ontogeneticevents, the majority-rule method (Yeh 2002),and Ontogenetic Sequence Analysis (OSA;Colbert 1999; Colbert and Rowe 2008). Eachmethod yielded separate hypotheses of onto-genetic sequence that subsequently wereevaluated for congruence, individual ontoge-netic variation (polymorphism), and the ef-fects of preservational bias and sample-size.Throughout, the statement ‘‘ontogeneticevent’’ refers to the development and appear-ance of a single morphological structure. ForM. pelikani and H. longicostatum, those eventsare ossification events where new bones haveossified. The term ‘‘stage’’ refers to a particularlevel of morphological maturity that is distin-guished from other such developmental stepsby the suite of structures present. During eachsubsequent stage one or more new ontogenet-ic events have occurred.

Ontogenetic Sequence Reconstruction

Size-Based Methods.—Using size as a proxyfor age or maturity, I generated separaterankings based on skull length and trunklength. In developmental studies based onvertebrate fossils, it is common to use skulllength as the standard for comparison in placeof body length when the two measurementsare statistically correlated (e.g., Witzmann andPfretzschner 2003). In some cases, moreindividuals are preserved with a skull thanwith a complete trunk, and therefore use ofskull length can increase the available samplesize (e.g., Witzmann and Pfretzschner 2003).However, the assumption that skull lengthand body length are interchangeable andwould provide the same ontogenetic sequencereconstruction had not been tested previously.Therefore, from each measurement I generat-ed a separate ossification sequence in order toevaluate congruence. Skull length provides alarger sample size (n ¼ 60, M. pelikani; n ¼ 11,H. longicostatum) than trunk length (n ¼ 40,Microbrachis; n ¼ 10 Hyloplesion). Skull lengthwas measured along the midline from the tip

of the snout to the posterior margin of thepostparietals. Trunk length was taken from theanterior end of the atlas to the anterior end ofthe first sacral vertebra. The only specimensexcluded were those for which length mea-surements could not be obtained because ofpoor preservation.

Increasing Number of Ontogenetic Events.—One size-independent method of sequencereconstruction ranks individuals according toincreasing number of bones or other morpho-logical structures (e.g., Maisano 2002a,b). Thenumber of ossifications present in an individ-ual, for example, should be a proxy formaturity or ‘‘ontogenetic advancement’’(Schoch 2002) because as development pro-gresses more elements become ossified and itis unlikely that structures would be resorbedlater. However, this technique is more straight-forward for extant taxa than for extinct onesbecause of the uncertainty produced by poorfossil preservation. In neontological studies,ranking begins with the specimen exhibitingthe fewest elements, taken to demonstrate theearliest stage of development (Maisano2002a,b). This is not always clear in paleonto-logical studies because a specimen maypossess fewer structures as a result of poorpreservation rather than early maturity. Forexample, some specimens may show only theanterior portion of the body or are preservedonly in dorsal view. As a result, purelynumerical criteria (e.g., bone counts) couldnot be used alone and size had to be used incombination with increasing number of onto-genetic events.

The total sample for each taxon first wasordered according to skull and/or bodylength (not all specimens had both available),then manually ranked to reflect the appear-ance of ossified elements, beginning with theindividuals exhibiting the fewest ossifications.For example, if a given specimen possesses anossified humerus, radius, and ulna, then thenext, developmentally more advanced indi-vidual in the hierarchy would be expected tohave those elements plus additional ossifica-tions. Where specimens provided redundantinformation because they possessed the samesuite of ossified elements, their placementrelative to one another does not influence the


relative order of ossification. If no length wasavailable and poor preservation was an issue,placement was based on the combination ofelements that were positively present. Forexample, if a block of sediment containedonly the posterior portion of an individual (A),and that specimen positively possessed alllong bones, the ilium, and the ischium, then(A) was placed later in the series than a morecomplete specimen (B) with an ilium but notan ischium even though (B) presents moreossifications. An assumption is made aboutthe maturity level of (A), but the total pool ofindividuals is available to resolve the ontoge-netic sequence.

Majority Rule.—Because of problems withthe assumption that size and maturity arecorrelated, I included a method based on themajority-rule ranking proposed by Yeh (2002),which is independent of size. In this tech-nique, a ‘‘majority-rule’’ (modal) sequence isconstructed from a count of the number ofspecimens that possess each separate element.For example, if the radius is present in 20individuals and the ulna in 25, then it isinferred that the ulna ossified first becausebones that form earlier will be present in moreindividuals. The advantage of this method isthat it provides an objective, size-independentranking. However, the technique is moresuitable for extant specimens than for fossilsbecause of poor preservation in the latter. It iscommon for fossil specimens to be missinglimbs or portions of the skull or trunk, andtechniques based on bone counts alone cannotincorporate information provided by thecombination or suites of elements present indifferent individuals. In order to account forthe effect of preservation, bone counts (totalpositive events for each element) were firstcompiled according to the method proposedby Yeh (2002) and then subsequently adjustedby multiplying the total number of positiveevents by the ratio of missing events topositive events and adding the result to thenumber of positive events. Both the unadjust-ed and weighted values were then used toorder the appearance of bones, starting withthe element that received the highest score. Allavailable specimens of M. pelikani and H.longicostatum were used in this analysis.

Ontogenetic Sequence Analysis.—OSA is amethod that was developed not only tofacilitate sequence reconstruction but also toexplore and account for polymorphism (Col-bert 1999; Colbert and Rowe 2008). OSAdiffers fundamentally from the other methodsof sequence reconstruction because by pre-dicting all equally parsimonious sequences, itprovides the full range of complete sequencesthat potentially could be expressed in apopulation. Using the other methods ofreconstruction, the observed sequences re-corded for each specimen are only fragmentsof complete ontogenetic trajectories becausethe sequences terminate at the stage when theanimal died. Potential alternative ontogeneticsequences cannot be estimated, and measure-ment of polymorphism can be done onlyretroactively by surveying the sample foraberrant ontogenetic events relative to thesingle, reconstructed sequence. On the otherhand, OSA provides statistics and measure-ments so that in addition to the identificationof polymorphic individuals and highly vari-able elements, the likelihood of alternative,complete ontogenetic sequences can be calcu-lated and those sequences can be compareddirectly with one another.

Based on the principle of parsimony, OSAuses observed data to construct a map of allpossible equally parsimonious developmentalpathways. In short, cladistic methods are usedto optimize character changes, in this casedevelopmental events, onto trees constructedfrom the developmental data. The trees areused to build reticulating diagrams thatrepresent the predicted sequence pathways.Under OSA, because all developmental path-ways reticulate, eventually connecting theleast mature condition to the most mature,all procedures are carried out from leastmature to most mature (normal treatment)and then separately from most mature to leastmature (reverse treatment). Methods followthose outlined by Colbert (1999), althoughsome details were modified in order to dealwith the large amount of missing data infossils. The sample size for H. longicostatumwas too small for OSA; only M. pelikani wasanalyzed.


In OSA, a table containing the presence/absence states for individual elements for thetotal sample first is compiled into a datamatrix similar to those used in cladisticanalyses. Specimens are treated as operationaltaxonomic units (OTUs) and developmentalevents are scored as characters. Bones thatdefinitively are absent are coded as (0), thoseossified as (1), and missing information as (?).Transformation of the epipodials was treatedas a multistate character with absence as (0), asingle ossification as (1) and multiple carpalsor tarsals coded as (2).

The next step in OSA is to identifyindividuals that are redundant because theyexhibit the same suite of ontogenetic events. Asingle specimen is chosen from each redun-dant batch to represent the unique combina-tion of events present in that group ofindividuals. The chosen specimens are treatedas representative ‘‘semaphoronts’’ and allother redundant individuals are culled fromthe matrix. The number of culled individualssupporting each representative semaphorontis recorded. In studies using extant taxa thisprocess is straightforward. However, in pale-ontological studies, the prevalence of missingdata means that the redundant individualsmay match up with multiple representativesemaphoronts when gaps in the partial se-quences are considered. In my analysis, if aredundant individual matched more than onesemaphoront, the frequency support from thatindividual was divided among the multiple,supported semaphoronts. For example, if anindividual was redundant for four semaphor-onts, that individual contributed 0.25 towardthe total frequency support calculated for eachof those four semaphoronts. If a culledindividual supported only a single semaphor-ont, then that individual contributed a valueof 1.0 toward the frequency of the semaphor-ont. The frequencies are used later to deter-mine the modal developmental sequence (orsequences), interpreted to be the one mostoften expressed in the population, and toproduce likelihood values for all mappedsequences. Culling resulted in 13 M. pelikanisemaphoronts available for analysis.

In paleontological studies it is rare to havespecimens showing all least mature states

(e.g., no bones ossified) or all most maturestates (e.g., all bones ossified). Under standardOSA, that problem is circumvented somewhatbecause a hypothetical least mature individu-al, coded with all (0)s, is added to the matrixbefore the analysis is begun. In addition, toprepare the reverse treatment, the matrix iscopied and states are re-coded in reverse. Inother words, for binary characters, the maturestate (present) becomes (0) and the leastmature state (absent) becomes (1). Similarly,for my study, multistate characters originallyrepresented as (0), (1), (2) were changed to (2),(1), (0), respectively. Finally, the reversetreatment matrix requires a hypothetical mostmature individual, which is given all (0)s toreflect maturity under the new coding scheme.

Two separate analyses were run in PAUP*(4.0b10; Swofford 2002) as a heuristic searchusing parsimony with tree bisection-reconnec-tion (TBR). In OSA characters are treated asunweighted, but designated irreversible ‘‘up’’(normal) because even if elements are re-sorbed later in ontogeny, those events wouldbe further transformational steps. Only treeswith the ‘‘best score’’ from each analysis weresaved for use in MacClade (version 3.01;Maddison and Maddison 1992). The treesand corresponding matrices were opened inthat program and the ‘‘trace all characters’’function was implemented with both ambig-uous and unambiguous changes reflected asbranch lengths. All trees were inspectedmanually and the reticulating diagram wasdrawn following methods described by Col-bert (1999). Each partial sequence segment isshown on the map as a single bold lineconnecting a path between two semaphoronts.If multiple character changes occur and theorder is not resolved, the path length must beproportional to the number of changes. Inaddition, each complete pathway from leastmature to most mature must contain transi-tions for all characters and state changesscored. Many of the trees differ in characteroptimization only (ACCTRAN and DEL-TRAN), which results from the large amountof missing data in fossils and the standardway PAUP* handles missing informationunder parsimony (i.e., ignores missing statesduring reconstruction, but assigns the most


parsimonious state after OTU placement).Those situations are reflected in the map aspathways with double lines connecting mul-tiple instances of the same semaphoront. Byconvention, the modal sequence, which isdesignated by the highest frequency support,is used as the spine for the two-dimensionaldrawing. Any pathway that differs from themodal sequence is considered polymorphic.Semaphoronts (and their supporting redun-dant specimens) are assigned a range ofpossible maturity stages, and developmentalevents are ranked according to the averagesequence position occupied among all predict-ed sequences, equivalent to an ‘‘average’’ontogenetic sequence (Colbert 1999).

Ontogenetic Sequence Variation

Polymorphism occurs when an individualexhibits an alternative ontogenetic sequencerelative to those of others in the population. Inmy evaluation, strict polymorphism is identi-fied when an individual exhibits an ‘‘out-of-order’’ sequence of ossification relative to themodal sequence reconstructed for the sample.In other words, the samples were assessed forspecimens that show ossification of oneelement while definitively lacking ossificationof a bone that is predicted to appear earlier inontogeny, based on the reconstructed se-quence. However, a difference in size at aparticular maturity level does not equate tosequence variation. Those individuals do notdemonstrate an ‘‘out-of-order’’ sequence ofossification; bone A still precedes the unde-veloped bone B, even though B might beanticipated in a specimen of such large size.Instead, these bigger animals are at the samestage of growth as others that are measurablysmaller, and vice versa.

A second type of polymorphism resultswhen a specimen is lacking an element thatwas reconstructed as appearing at the sametime as other elements that are clearly ossifiedin the individual. Simultaneous appearancecan be produced in two ways. The first occurswhen a single specimen exhibits the firstappearance of multiple elements. In manycases, especially in paleontological studies,those multiple first occurrences happen be-cause intermediate stages of ossification that

could break up the apparent simultaneity arenot preserved. In other words, the sequence isunresolved. The second type of simultaneousappearance also stems from uncertainty, butoccurs specifically when multiple individualspresent conflicting evidence for the elementsthat ossify during a particular stage ofdevelopment. When size methods are used,for example, conflict occurs when specimensof the same length demonstrate the presenceof different elements. In my evaluation,because order is indistinguishable, those ossi-fication events conservatively are presented aselements that ossify simultaneously, or at thesame stage. Simultaneity was applied underthe ‘‘increasing number of ontogenetic events’’method when order was ambiguous becausespecimens with evidence for the next stage ofossification demonstrated the subsequent ap-pearance of different bones (i.e., both have ahumerus, radius, and ulna, but in addition onehas a femur and another has a cleithrum).Elements that have the same count when themajority-rule consensus method is used alsowere identified as simultaneous appearance.Under OSA, order is unresolved when asequence segment connecting two semaphor-onts represents more than one charactertransition. Whether the simultaneity is agenuine phenomenon or is instead an artifactof poor resolution, in situations where aspecimen is lacking one of the simultaneouselements, that individual exhibits variationrelative to the sequence reconstructed for thesample.

The amount of polymorphism is quantifiedand compared by using a number called thedisparity estimate (Maisano 2002c). The dis-parity estimate is a measure of sequencevariation given by the ratio between ontoge-netic events that have not yet occurred(because of polymorphism) to the total num-ber of definitive ontogenetic events. To calcu-late that number I compared the ossificationsequence of each individual to the reconstruct-ed sequence derived from each method. I usedboth strict and simultaneous polymorphismsin the calculation. Larger specimens with thesame ossification sequence as smaller speci-mens were considered to be at the same stageof maturity as the smaller individuals and,


therefore, not to exhibit polymorphism. Donein this way, the calculation is more similar tocalculations of the disparity estimate ofMaisano (2002a,b) for specimens ordered byincreasing number of ontogenetic events thanfor those ordered by length, because in thesize-ordered studies of Maisano (2002a,b), alllarger specimens expected to demonstrate anossification event but missing that elementwere included in the calculation. The numberof individuals demonstrating polymorphismis reported for each reconstruction method.

Institutional Abbreviations

AMNH – American Museum of NaturalHistory, New York, New York; CGH –Narodni Museum, (now National MuseumPrague), Prague, Czech Republic; M – Nation-al Museum Prague, (previously NarodniMuseum), Prague, Czech Republic; MB –Museum fur Naturkunde, Humboldt Univer-sity, Berlin, Germany; NHMW – Naturhistor-isches Museum, Vienna, Austria; RSM – RoyalScottish Museum, Edinburgh, U.K.; St �Narodni Museum, (now National MuseumPrague), Prague, Czech Republic.

Results

Ossification Sequence of Microbrachis pelikani –Skull Length

Skull, Scales, Branchial Plates, ScleroticRing.—All specimens have a completely ossi-fied skull with a complete circumorbital seriesand an ossified braincase. Dermal scales areconsistently present in all specimens exam-ined. The presence or absence of branchialplates in M. pelikani has been a point ofcontention since the first report of suchelements by Carroll and Gaskill (1978). Iobserved definitive branchial plates in seven(9%) of the 77 M. pelikani specimens examined(Supplementary Table 1). Because the platesare found in individuals with skull lengthsranging from 10 to 22 mm, I inferred that theyare present in individuals of most growthstages but are affected greatly by taphonomy.Branchial plates potentially are unossified inthe smallest individuals, but poor preserva-tion cannot be ruled out.

Similar to the branchial plates, the scleralossicles that form the sclerotic ring also areprone to poor preservation, and many speci-mens lack these structures entirely. Althoughpreservation always is incomplete, ossifiedscleral ossicles are present in 18 individuals(23%), the smallest with a skull length of 10mm (Supplementary Table 1). No part of ascleral ring or ossicle impression could belocated in the three smallest specimens,suggesting that the ossicles were unossifiedin the earliest stages of development, althoughone of those three (CGH267, 8.2 mm) waspreserved in ventral view, making it unlikelythat any ossicles would be visible even ifpresent. Additionally, the poor preservationpotential of the scleral ossicles does not ruleout the possibility of ossification in smallerindividuals.

Vertebral Column.—Fully ossified neuralarches and vertebral centra were observed inall specimens of M. pelikani examined, includ-ing the smallest known individual (skulllength 7 mm). Ribs are also present in allindividuals. Haemal arches are identifiedmost easily in specimens preserved in lateralview with little disturbance in the tail region,and are present in 12 (36%) of the 33individuals that possess associated series ofcaudal vertebrae (Supplementary Table 1).Skull lengths of the individuals with haemalarches range from 10 to 20 mm, although aswith branchial plates, it is possible that thesestructures were lost or weakly ossified insmaller individuals.

Limbs and Girdles.—No individuals defini-tively lack any long bones, but that is a resultof poor preservation rather than early devel-opmental stage of the specimens (Supplemen-tary Table 1). Some specimens are missingsections of skeleton because of broken slabs orloss of thin layers of coal (marked as ‘‘.’’ intables) whereas others were too badly pre-served to allow for positive identification ofelements (denoted by ‘‘?’’ in tables). In thesmallest specimen examined (NHMW1983_32_66; 7 mm skull length; Supplementa-ry Table 2), the humerus, radius, ulna, andfemur unambiguously are present. Unfortu-nately, in both that individual and the nextsmallest animal (8.2 mm skull length), the


layer of coal containing the specimen ends justanterior to the area that would have preservedthe pelvic girdle (the single femur of NHMW1983_32_66 is anteriorly displaced). Positiveidentification of the tibia and fibula is firstfound in a specimen with a skull length of 9.4mm, along with the first appearance of anossified clavicle and ilium (Fig. 2). The 9.4-mmspecimen also possesses an ossified radius,ulna, humerus, and femur, confirming earlyossification of those elements in the smallerspecimens. With the exception of the scapula,the remaining girdle elements also ossifyrelatively early and in quick succession. Anossified interclavicle and ischium are found ina specimen with a skull length of 10 mm, andthe first appearance of a pubis (poorlyossified) occurs in an animal with a skulllength of 11 mm. The scapula is not present inany specimen of M. pelikani with a skull lengthless than 17 mm.

Carpals, Tarsals, Metapodials, and Phalanges.—All specimens of M. pelikani that I examinedhave ossified phalanges and metapodials,including the three smallest specimens. Car-pals and tarsals, however, ossify relatively latein ontogeny, a common pattern across Tetra-poda. The tarsals ossify before the carpals; asingle proximal tarsal is first found in aspecimen with a 16-mm skull length (Supple-mentary Table 2). Ossification of the carpalsfollows soon after, with a single proximalcarpal ossified in an animal with a skull lengthof 17 mm. One specimen with a skull length ofmore than 20 mm has three ossified tarsals,and one of the largest specimens, with a skulllength of 28 mm (CGH3018), possesses fivedistinct carpals.

Ossification Sequence of Microbrachis pelikani –Trunk Length

The two length-based subsamples of M.pelikani do not overlap completely; 35 speci-mens are shared between them. The resultingossification sequences generally agree in therelative order in which elements first appear(Fig. 2). However, when ranked according totrunk length, there is apparent ossification ofthe clavicle, ilium, ischium, and all long bonesin the smallest specimen of the trunk lengthsample (45 mm; Supplementary Table 3).

There are no smaller individuals that record

earlier stages in development that might show

stepwise appearance of those elements (i.e.,allow finer resolution in sequence). The

interclavicle is the next element to ossify

(trunk length 47 mm) followed by the pubis

(trunk length 52 mm). Ossification of the

scapula and first tarsal (proximal row) isunresolved because both appear first in an

individual with a trunk length of 69 mm. The

first carpal (proximal row) ossifies later in a

FIGURE 2. Chart showing the first appearance of ossifiedelements of the postcranium in Microbrachis pelikani,ordered according to increasing skull length, left, andbody length, right. Abbreviations: car, carpal; cla, clavicle;clei, cleithrum; fem, femur; fib, fibula; hum, humerus; ili,ilium; intcl, interclavicle; isch, ischium; pub, pubis; rad,radius; sca, scapula; tar, tarsal; tib, tibia; uln, ulna.


specimen with a trunk length of 73 mm. Noneof the specimens in the trunk length sampleexhibit multiple ossified carpals, but oneindividual with a trunk length of 120 mmpossesses three ossified tarsals.

Ossification Sequence of Microbrachis pelikani –Increasing Number of Ontogenetic Events

The least mature specimen demonstratesossification of the humerus, radius, and ulna(Fig. 3, Supplementary Table 1). The posteriorpart of that specimen is not preserved, but it isclearly missing the clavicle, which is present inindividuals with more ossifications. The nexttwo informative individuals both show evi-dence of an ossified femur, in addition to theforelimb elements found in the least maturespecimen. However, because of missing dataresulting from poor preservation, the twoprovide conflicting evidence regarding addi-tional new elements that ossify with thefemur. One shows the additional presence ofthe ilium, tibia, fibula, and clavicle whilemissing information about the cleithrum, andthe second shows the positive presence of acleithrum while missing information about theilium, tibia, fibula, and clavicle. More maturespecimens exhibit all of those ossifications andthus, in the absence of new data, the femur,tibia, fibula, ilium, clavicle, and cleithrum arereconstructed as appearing simultaneously.The same ambiguity affects the interclavicleand ischium, which also are reconstructed asossifying together in the next stage. The pubisossifies after the ischium and interclavicle.

The order of appearance of the tarsals,carpals, and scapula also was ambiguousbecause specimens presented conflicting in-formation. For example, specimens exhibitingthe first potential ossification of the scapulalacked all epipodials and those exhibiting thefirst potential ossification of the tarsals lackedcarpals and scapulae. However, given thenumber of specimens that clearly have eachof the ambiguous elements, as well as the sizeof the individuals with conflicting informa-tion, parsimony suggests an order of scapula,tarsals, then carpals. The uncertainty is repre-sented in Figure 3 by listing the elements asossifying during the same stage, but offsettingeach bone to reflect the suggested order.

The ossification sequence derived from

ranking by increasing number of ontogenetic

events in M. pelikani also is consistent with the

two length-based methods. Even with the

clustering produced, the scapula is suggested

to ossify in association with the epipodials and

after the pubis, the pubis appears after the

ischium, and the remainder of the girdle and

limb elements ossify earlier in ontogeny. The

appearance of the limb bones is less clustered

than in the trunk sequence, similar to that

constructed from skull length.

FIGURE 3. Chart showing the first appearance of ossifiedelements of the postcranium in Microbrachis pelikani,ordered according to increasing number of ontogeneticevents, left, and majority-rule method, right. Abbrevia-tions as in Figure 2.


Ossification Sequence of Microbrachis pelikani –Majority Rule

The majority-rule ranking proposed by Yeh(2002) resulted in a high degree of ossificationevent resolution; there is only a single occur-rence of simultaneous events. Subsequentadjustment for preservation yielded onlysingular events (Fig. 3, Supplementary Table4). Using the unadjusted results, the humerusis the first element to ossify, followed by theclavicle and radius together and then the ulna.Ossification of the cleithrum occurs next,followed by the femur, and then the intercla-vicle. The ilium ossifies next, and the tibia,fibula, ischium, and pubis all ossify prior tothe scapula, in that order. As with the othermethods, the epipodials are the final elementsto ossify; tarsals precede the carpals. Whenweighted to account for preservation, theresults are similar, although the clavicleprecedes the radius in a separate ossificationstage and the scapula appears before thepubis. Although more finely resolved, themajority-rule method is consistent with thereconstructions reported above in that thelimb bones and dermal girdle elements ossifyrelatively early, the scapula ossifies rather late(after the ilium and ischium), and the pubisossifies early, before the epipodials.

Ossification Sequence of Microbrachis pelikani –Ontogenetic Sequence Analysis

OSA resulted in a total of 584 trees underthe ‘‘normal’’ treatment and 56 under the‘‘reverse’’ treatment. The reticulating map ofpolymorphic sequences (Fig. 4) exhibits fewunresolved sequence segments and predicts610 potential sequences (Supplementary Ap-pendix 1). Nearly all sequences involvemissing data, which frequently are manifestedas pathways with double lines signifyingalternative optimizations (Fig. 4). In realitythe map is three-dimensional, and semaphor-onts with a lot of missing data, such as S3(CGH 251) tend to fill in the space aroundpathways constrained by unambiguous data.Additionally, the larger the amount of missingdata, the greater the number of alternativepathways connected to the semaphoront, andthe greater the range of maturity stages thatthe semaphoront could be hypothesized to

occupy. This is exemplified by S3, which couldoccupy any relative maturity level from 5 to 12and contains more than 50% missing data(Table 1). Despite the large amount of missinginformation, S3 is informative because itconstrains pathways early in the ossificationsequence. In other words, even though muchof the information is missing, the patternproduced is not random.

Additionally, if missing data were ignored,events 5, 6, 7, 9, 12, 13, 14 (long bones andilium) would be viewed as uninformativebecause, except for differences in missingdata, these bones are ossified in all specimensof M. pelikani. In fact, in standard phylogeneticanalyses, PAUP* considers those to be unin-formative characters. However, when the‘‘normal’’ and ‘‘reverse’’ treatments are inte-grated to form the reticulum, and partly as theresult of the way PAUP* handles missing data,the combination of missing and presentinformation in semaphoronts S1 and S3contributes a large amount of early sequenceresolution. Many of those seven ‘‘uninforma-tive’’ events are mapped as unresolved pairs,but overall the block is broken up intoresolved segments. Still, the missing data dolead to many distinct optimizations early indevelopment, which in turn nearly triple thenumber of potential polymorphic sequencesthat would have been predicted had therebeen less missing information. In other words,those numerous distinct optimizations areartifacts of missing data that reflect uncertain-ty because many different starting sequencesare equally parsimonious.

A total of 12 predicted sequences share amaximum frequency support value of 55.89(Supplementary Appendix 1) and are consid-ered to be modal sequences. The modalsequences generally are congruent with oneanother, differing mainly in the sequenceposition of a single ossified carpal relative tomultiple carpals and tarsals, the cleithrumrelative to the tibia and fibula, and the ulnarelative to the femur and ilium. Relationshipsthat are invariant among the 12 modalsequences include ossification of the humerusand radius together before all other elements,ossification of the ilium and femur togetherbefore all remaining elements except for the


FIGURE 4. Reticulating diagram of OSA for Microbrachis pelikani. Representative semaphoronts (nodes) are depicted asovals where the horizontal length is proportional to frequency support. Single bold lines indicate sequence segments(paths) observed on trees. Double lines represent different optimizations caused by missing data. Character transitionsare given alongside the path on which they occur. Characters are listed in Table 1. Note that one node could be eithersemaphoront 9 or 13 in different polymorphic sequence reconstructions. Figure provided in color in online edition.


ulna, and appearance of the tibia and fibulatogether before the clavicle, interclavicle,ischium, pubis, scapula, and epipodials. Ad-ditionally, the modal sequences agree in thewell-resolved appearance of the clavicle, is-chium, interclavicle, scapula, and single tarsal,in that order. Overall the modal patterns aresimilar to the ossification sequences producedby the other reconstruction methods, althoughthe early stages are better resolved with OSA(perhaps artifactually because of missing data)than they were with the methods based onlength or increasing number of ontogeneticevents.

The average sequence, calculated from thesequence positions found in all 610 alterna-tives, also was reconstructed and is congruentwith the modal sequences (SupplementaryAppendix 2; Figs. 5, 6). The average sequencepredicts that the humerus and radius firstappear together, then the ilium and femurtogether, followed by the ulna. Next, the tibiaand fibula ossify as a pair, and then thecleithrum, ischium, clavicle, interclavicle, andpubis all appear separately and in that order.Finally, a single proximal tarsal ossifies,followed by a single proximal carpal, thescapula, multiple tarsals, and then multiplecarpals. Despite differences in the resolution ofossification events and details about thesequence position of dermal girdle elements,both the modal and average OSA ossificationsequences are consistent with the previousmethods in suggesting early appearance of the

long bones and ilium, late ossification of thescapula, and relatively early formation of thepubis.

Ossification Sequence of Hyloplesionlongicostatum

The rankings established for H. longicosta-tum, presented in Supplementary Tables 5�8,have an almost complete overlap of individ-uals, which is reflected in the strong congru-ence among the resulting ossificationsequences (Figs. 7, 8). The two length-deriveddata sets differ only in the timing of appear-ance of multiple tarsals. The ossificationsequence constructed from increasing numberof ontogenetic events, however, presentedmore challenges in its formulation. As withsome ossification events in M. pelikani, theappearance of many elements is ambiguousbecause multiple specimens exhibit the samenumber of new ossifications but missing dataprevent a clear establishment of order. Whenorder was indecipherable, elements weredepicted as ossifying simultaneously (i.e.,unresolved). The result is a clustering ofevents into fewer separate stages, but therelative order of ossification is consistent withthe length-derived samples. The majority-ruleranking for H. longicostatum also presentedsimultaneous ossification events, althoughfewer than with the other methods (Fig. 8).One major difference in sequence using thatmethod is the ossification of all hindlimb longbones before any of the forelimb bones.

TABLE 1. Representative semaphoronts of Microbrachis pelikani utilized in OSA. Numbers in parentheses represent thecharacter numbers used in the analysis. Abbreviations: car, carpals; clav, clavicle; cleith, cleithrum; fem, femur; fib, fibula;hum, humerus; ili, ilium; intcl, interclavicle; isch, ischium; pub, pubis; rad, radius; sca, scapula; tar, tarsals; tib, tibia; uln,ulna.

Specimensca(1)

cleith(2)

clav(3)

intcl(4)

hum(5)

rad(6)

uln(7)

car(8A,B)

ili(9)

isch(10)

pub(11)

fem(12)

tib(13)

fib(14)

tar(15A,B)

CGH267 0 0 0 ? 1 1 1 0 ? ? ? ? ? ? ?St190 0 ? 1 0 1 1 1 0 1 0 0 1 1 1 0CGH251 0 1 0 ? 1 1 ? ? 1 ? ? 1 ? ? ?NHMW1898_x_29 0 1 1 1 1 1 1 0 1 1 0 1 1 1 0St193 0 1 1 ? 1 1 1 0 1 1 1 1 1 1 0CGH5 0 0 1 1 ? 1 1 0 1 1 1 1 1 1 1CGH2098 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1CGH254 0 1 1 0 1 1 1 1 1 ? ? 1 1 1 ?CGH727 0 1 1 1 1 1 1 1 ? ? ? ? ? ? ?M1694 1 1 1 1 1 1 1 0 1 1 1 1 1 1 0MB.Am.810.1–2 1 1 1 ? 1 1 1 0 1 1 1 1 1 1 1MB.Am.822.1 ¼MB.Am.17 1 1 1 1 1 1 1 2 1 ? ? ? ? ? ?St116 ? 1 1 1 1 ? ? ? 1 1 1 1 1 1 2


Additionally, the scapula and interclavicle are

reconstructed as ossifying after multiple tar-

sals, a sequence not reported by any other

method. Distribution of the branchial plates,

haemal arches, and sclerotic ring is provided

in Supplementary Table 5. OSA could not be

used for H. longicostatum because of low

sample size.

Intraspecific Variation

Ten specimens from the M. pelikani skull-

length sample exhibit an out-of-order ossifica-

tion sequence, representing 16.1% of thatsubset (Table 2, Supplementary Table 2). Thedisparity estimate is 3.1%, which falls into thelower range of figures reported for ossificationsequences of extant taxa (Maisano 2002a,b;Maxwell 2008). The estimate probably isconservative because poor preservation mightobscure additional variations. Most of thedisparity is related to the appearance of thescapula and epipodials, affecting eight of theten individuals, and variation is not alwaysindependent, because multiple specimensshow sequence variation for multiple ele-ments. Ten instances of polymorphism aresimultaneous, where an element is unossifiedeven though elements that appear at the samestage are present. Four cases of strict poly-morphism occur when an element that shouldbe ossified has not yet appeared even thoughelements that ossify at later stages are defin-itively present.

Seven individuals of the trunk-length dataset, representing 17.1% of the total, showvariation in ossification sequence. However,the disparity estimate is slightly lower (2.5%)with three cases of strict polymorphism andsix of simultaneous events (Table 2, Supple-mentary Table 3). When ordered by increasingnumber of ontogenetic events, the disparityestimate for M. pelikani reaches a maximumvalue of 3.2%. However, the disparity isreduced to a low of 1.1% if the weaklysupported order of scapula, tarsals, andcarpals is accepted. Treating those elementsas ossifying during the same stage results in 11polymorphic individuals (13.9% of the speci-mens; Table 2, Supplementary Table 1). Themajority-rule method resulted in a smallpercentage of polymorphic individuals(8.9%) and a low disparity estimate (1.6%),with seven strict polymorphisms (Table 2,Supplementary Table 1). OSA provided adisparity estimate (2.0%) in line with those ofother methods when the average sequence iscompared with all observed specimens of M.pelikani (Table 2). Only seven individuals(9.1%) exhibit an ossification sequence incon-gruent with the average sequence (Supple-mentary Table 1). However, the average OSAsequence gives the highest quantity of strict

FIGURE 5. Chart showing the first appearance of ossifiedelements of the postcranium in Microbrachis pelikani,ordered according to the average sequence reconstructedby OSA. Abbreviations as in Figure 2.


polymorphisms (11) and does not identify anysimultaneous polymorphisms.

In addition to identifying polymorphicindividuals and ossification events, OSA canbe used to inspect common (i.e., conserved)patterns more deeply. According to the aver-age sequence positions for all ossificationevents across all predicted ossification se-quences, appearance of the humerus andradius before all other elements is invariant(100% of 610; Tables 3, 4). Other invariantrelationships include the ulna before the tibia,fibula, ischium, interclavicle, clavicle, pubis,scapula, and epipodials (100%); the iliumbefore the tibia, fibula, cleithrum, ischium,interclavicle, clavicle, pubis, scapula, andepipodials (100%); the femur before the tibia,fibula, cleithrum, ischium, interclavicle, clavi-cle, pubis, scapula, and epipodials; the tibiaand fibula are never resolved relative to oneanother but always ossify before the ischium,interclavicle, clavicle, pubis, scapula, andepipodials (100%); the cleithrum before theepipodials and scapula (100%); the ischium

before the pubis, first ossified tarsal, scapula,and multiple epipodials (100%); the intercla-vicle before the pubis, first ossified tarsal,scapula, and multiple epipodials (100%); theclavicle before the scapula, and multipleepipodials (100%); the pubis before scapulaand multiple epipodials (100%) as well as asingle tarsal (97%); and the scapula beforemultiple carpals (100%).

The cleithrum is the most variable elementin terms of sequence position, exhibiting thehighest standard deviation (2.56) and thelargest range (5�13). The ossification of thefirst carpal also is highly variable, spanningsequence positions 9�16 (standard deviation1.72). However, the earliest potential ossifica-tion of the first carpal (position 9) occurs inonly 15/610 sequences (2.5%), and is associ-ated directly with missing data, suggestingthat early ossification is unlikely to be ob-served in the population.

The disparity estimates for H. longicostatumwere generally lower than for M. pelikani(Table 2). The skull and trunk length methods

FIGURE 6. Graph depicting the average, minimum, and maximum sequence positions for postcranial ossification eventsof Microbrachis pelikani as reported by OSA. Events, given on the horizontal axis, are ordered are according to the modalsequence. The average sequence is represented by the line connecting the average sequence positions.


yielded estimates of 1.3% and 1.4%, respec-tively, while those based on increasing num-ber of ontogenetic events and the majority-rule method were calculated to be 2.3% and3.4%, respectively. Additionally, the skull- andtrunk-length samples each exhibited only asingle polymorphic individual (10�11.1%;Supplementary Tables 6, 7), whereas the othertwo methods each had two polymorphicspecimens, making up a relatively high16.7% of the data (Supplementary Table 5).The only strict polymorphism resulted fromthe trunk length sequence reconstruction(M1377; Supplementary Table 7).

Discussion

Methods of Ontogenetic SequenceReconstruction

The ontogenetic sequences that I establishedfrom each method are widely in agreementwith one another. That robustness stems fromthe fact that regardless of which method ofreconstruction is used, only a small number of

specimens out of the total sample are supply-ing information on the first appearance ofelements. Data from other individuals areredundant, ambiguous, or can aid in resolvingconflicts but do not determine the sequencedirectly. Frequently, the informative specimensare the same from method to method, even ifthere is a change in the ranking of individualspecimens relative to one another in terms ofinferred maturity level. Despite that underly-ing similarity, reconstructions were not repro-duced identically under the various methods,and results from the different methods werenot always affected in the same way by thepreservational bias inherent in paleontologicaldata.

Length-based methods allow for objectiveuse of all available specimens with measure-ments, although it is assumed that there is apositive correlation between size and maturi-ty. As with other methods, the specimensutilized for reconstruction can have a large

FIGURE 7. Chart showing the first appearance of ossifiedelements of the postcranium in Hyloplesion longicostatum,ordered according to increasing skull length, left, andbody length, right. Abbreviations as in Figure 2.

FIGURE 8. Chart showing the first appearance of ossifiedelements of the postcranium in Hyloplesion longicostatum,ordered according to increasing number of ontogeneticevents, left, and the majority-rule method, right. Abbre-viations as in Figure 2.


amount of missing data. However, unlikesome of the size-independent methods, rank-ing according to length does not necessitateassumptions or decisions about preservationand it is easy to visualize, compare, andinterpret bias caused by taphonomy becausereconstruction is based directly on the empir-ical data. Keeping preservation in mind, aslong as measurements are available, evenhighly fragmentary specimens can contributeto the sequence reconstruction because theirplacement relative to other individuals isknown, rather than inferred. Ordering isstraightforward and objective because the firstoccurrence of each ontogenetic event is de-rived independently from the smallest speci-men in which it appears. When the sequence isestablished by using size as a proxy for age orstage, the results are effectually describing theearliest size at which a taxon is capable ofproducing a specific element, not the size atwhich the structure develops in all individu-als. Perceptions about the size at which certainontogenetic events occur still are affected bypreservational bias; data may be missing forsmaller individuals. As with all other methodsused to reconstruct sequences, the resultingorder is only a hypothesis of the modalsequence for the population. In other words,it is the sequence that is expected to beexpressed by the largest number of individu-als although it will not be the only sequencepresent in the population. Furthermore, eventhough length is used to rank the specimens,size is not directly incorporated into thesequence, which remains a relative sequenceof the appearance of structures. Only therelative order of appearance, not the absolutetiming, is important within a comparativecontext.

A method based purely on the number ofmorphological structures present in eachindividual can provide a size-independentand objective reconstruction, but unfortunate-ly fossil preservation usually precludes use ofthis method in isolation. Nearly all of thespecimens exhibited missing data for at leastone postcranial element, and counts aloneignore the prevalence of missing information,which may bias the resulting sequence. Asmodified and implemented in my study,T

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however, the ‘‘increasing number of ontoge-netic events’’ method provides an advantagebecause it allows all available specimens to beused, whether or not length measurements areavailable, and permits incorporation of infor-mation from combinations of elements pre-sent. In addition, as with length-basedmethods, reconstruction is made directly fromempirical data. However, it also is affectedgreatly by the quality of preservation, which

can lead to subjective decisions. Conflicts aremost likely to occur when initiating theontogenetic sequence, because it can beunclear if the specimen with the fewestelements really is the least mature individualor if elements are missing because of poorpreservation. In my study, for example, strictlyusing the number of elements identifiesindividual CGH267 as the developmentallyearliest specimen, but that individual is

TABLE 3. Summary of M. pelikani ossification sequences from OSA in terms of a bone-by-bone comparison. Table shouldbe read horizontally as the number of sequences that have each row event occurring before each column event. Readingvertically gives the number of sequences that support each column event occurring before each row event. Numbers inparentheses represent the character numbers used in OSA. See Table 1 for abbreviations.

hum(5)

rad(6)

uln(7)

ili(9)

fem(12)

tib(13)

fib(14)

cleith(2)

isch(10)

intcl(4)

cla(3)

pub(11)

tar(15A)

car(8A)

sca(1)

multitar

(15B)

multicar(8B)

hum (5) x 0 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610rad (6) 0 x 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610uln (7) 0 0 x 271 271 610 610 542 610 610 610 610 610 610 610 610 610ili (9) 0 0 339 x 0 610 610 610 610 610 610 610 610 610 610 610 610fem (12) 0 0 339 0 x 610 610 610 610 610 610 610 610 610 610 610 610tib (13) 0 0 0 0 0 x 0 406 610 610 610 610 610 610 610 610 610fib (14) 0 0 0 0 0 0 x 406 610 610 610 610 610 610 610 610 610cleith (2) 0 0 68 0 0 204 204 x 388 430 340 502 562 610 610 610 610isch (10) 0 0 0 0 0 0 0 222 x 289 318 610 610 589 610 610 610intcl (4) 0 0 0 0 0 0 0 180 0 x 318 610 610 562 610 610 610cla (3) 0 0 0 0 0 0 0 270 289 292 x 479 554 580 610 610 610pub (11) 0 0 0 0 0 0 0 108 0 0 131 x 594 493 610 610 610tar (15A) 0 0 0 0 0 0 0 48 0 0 56 0 x 385 502 610 583car (8A) 0 0 0 0 0 0 0 0 21 48 30 117 225 x 350 469 581sca (1) 0 0 0 0 0 0 0 0 0 0 0 0 108 259 x 430 610multi tar (15B) 0 0 0 0 0 0 0 0 0 0 0 0 0 141 180 x 414

TABLE 4. Summary of M. pelikani ossification sequences from OSA in terms of a bone-by-bone comparison ofpercentages. Table should be read horizontally as the percentage of sequences that have each row event occurring beforeeach column event. Reading vertically gives the percentage of sequences that support each column event occurringbefore each row event. Numbers in parentheses represent the character numbers used in the analysis. See Table 1 forabbreviations.

hum(5)

rad(6)

uln(7)

ili(9)

fem(12)

tib(13)

fib(14)

cleith(2)

isch(10)

intcl(4)

clav(3)

pub(11)

tar(15A)

car(8A)

sca(1)

multitar

(15B)

multicar(8B)

hum (5) x 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100rad (6) 0 x 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100uln (7) 0 0 x 44 44 100 100 89 100 100 100 100 100 100 100 100 100ili (9) 0 0 56 x 0 100 100 100 100 100 100 100 100 100 100 100 100fem (12) 0 0 56 0 x 100 100 100 100 100 100 100 100 100 100 100 100tib (13) 0 0 0 0 0 x 0 67 100 100 100 100 100 100 100 100 100fib (14) 0 0 0 0 0 0 x 67 100 100 100 100 100 100 100 100 100cleith (2) 0 0 11 0 0 33 33 x 64 70 56 82 92 100 100 100 100isch (10) 0 0 0 0 0 0 0 36 x 47 52 100 100 97 100 100 100intcl (4) 0 0 0 0 0 0 0 30 0 x 52 100 100 92 100 100 100cla (3) 0 0 0 0 0 0 0 44 47 48 x 79 91 95 100 100 100pub (11) 0 0 0 0 0 0 0 18 0 0 21 x 97 81 100 100 100tar (15A) 0 0 0 0 0 0 0 8 0 0 9 0 x 63 82 100 96car (8A) 0 0 0 0 0 0 0 0 3 8 5 19 37 x 57 77 95sca (1) 0 0 0 0 0 0 0 0 0 0 0 0 18 42 x 70 100multi tar (15B) 0 0 0 0 0 0 0 0 0 0 0 0 0 23 30 x 68


missing the entire posterior half of the body.Lacking comparative information, and with-out invoking preformed assumptions aboutmaturity, there is no objective way to select theleast mature specimen. In order to circumventthe problem, it was necessary first to sort bysize to get an initial measure of maturity, andthen to manually adjust the rank of specimensto reflect maturity based on increasing num-ber of ontogenetic events and the combina-tions of structures present.

The majority-rule consensus method (Yeh2002) is effective for extant taxa but its poweris reduced for studies of fossils in which poorpreservation is a factor. Moreover, reconstruc-tion is based indirectly on empirical data.Adjusting for preservational bias did notchange the results other than to split up theonly simultaneous occurrence. Furthermore,swamping of ontogenetic data by taphonomicalterations may lead to illogical or weaklysupported reconstructions. The appearance ofthe femur, tibia, fibula, and ilium in H.longicostatum illustrates this effect well; ac-cording to the empirical data (SupplementaryTable 5), none of the specimens definitively aremissing any long bones. The reconstructedorder therefore is dependent entirely onmissing data resulting from poor preservation.

The majority-rule method, however, doesprovide a measure of the effect of preserva-tion, or perhaps the preservational potential,of the different elements. The results reflect thedegree to which each element individuallysuffers from preservational bias, and thereforemay be a useful metric in combination withtraditional length-based reconstructions. Thevalue obtained from multiplying the numberof positive occurrences by the fraction missingpredicts the number of additional positiveoccurrences that might have been present ifpreservational bias had affected all bonesequally. Elements that shift their relativesequence position between the unweightedand weighted treatments are more affected bypreservational bias. The majority-rule analysesalso reveal that for some elements, the amountof missing data from preservation is greaterthan the number of positive occurrences,which skews sequence reconstruction. Rela-tive patterns of preservational bias also can be

explored. In M. pelikani, the hindlimb exhibitsworse preservation than the forelimb and thegirdle elements are less well preserved thanthe limb bones. Carpals and tarsals showstrong preservational bias, too, which incombination with badly preserved girdleelements suggests that endochondral bonehas less preservational potential than dermalbone. It is unclear if the bias is caused bypersistence of weakly ossified endochondralelements throughout development, or simplythe late ossification of many endochondralbones, which reduces the chance of observa-tion in available fossils.

The most sophisticated and complex meth-od that I utilized, OSA, also is highlyobjective. Although for initial sequence map-ping OSA relies directly on only a smallnumber of the total pool of individuals, unlikethe other methods (with the exception ofmajority rule), the redundant specimens mak-ing up the majority of the sample are used toidentify the modal sequence and providefrequency support for sequence polymor-phisms. Calculation of frequency supportand the extrapolation of complete, polymor-phic sequences are themselves importantadvantages of OSA. The other methodsevaluated in my study are restricted to theconstruction of a single, complete sequence forthe population (a modal, or ‘‘typological’’sequence).

OSA also allows in-depth exploration ofpartial ontogenetic sequences, both predictedand observed, and can identify sequencesegments that are highly conserved amongalternative sequences. That information can beused in conjunction with traditional length-based methods to evaluate the likelihood ofdifferent segments of the modal sequenceconstructed from those data. For example,OSA demonstrates that the pubis almostalways precedes the epipodials, a result alsorecovered by the other reconstruction meth-ods. On the other hand, OSA shows thatpartial sequences involving the cleithrum aremore variable and that the sequence recon-structed according to skull length, where thecleithrum appears during the first stage of thedevelopment of the postcranial skeleton, is notpredicted by any of the 610 OSA sequences.


That result implies that the simultaneousrelationship reconstructed by using skull-length data probably is an artifact of poorresolution.

In addition to the modal sequence, OSA canprovide an average sequence calculated fromthe sequence position for each element pre-dicted by each polymorphic sequence. Be-cause the average sequence takes into accountall predicted sequences rather than relyingsolely on support from individual specimensthat might possess a large proportion ofmissing data, the average ontogenetic se-quence may be less affected by artifacts likepreservational bias than the modal sequence(Colbert 1999). Although OSA may be affectedby preservational bias because the fossils uponwhich it is based exhibit poor preservation,OSA incorporates the missing information,rather than ignoring it. Optimization ofmissing data may be complex and certainlyincreases the number of potential polymorphicsequences, but those estimates are a moreaccurate reflection of the uncertainty presentin the underlying fossils.

The importance of missing data is empha-sized in the first step of OSA, when sema-phoronts are selected and redundantspecimens are culled. In general, as theamount of missing data for an individualincreases, the number of representative sem-aphoronts that the specimen will match andsupport also increases. When specimens ex-hibit diametrically opposed character codingsbecause of missing data it may be difficult todecide if they should act as semaphoronts or ifthey should be culled. In my data set, becauseof missing information the only two individ-uals (S12 and S13) that provide partialsequences for mostly mature states, includingossification of multiple epipodials, potentiallycould have the same character coding (Table1). Indeed, these semaphoronts sometimes aremapped at the same node (Fig. 4). However,under different pathways, the two semaphor-onts clearly provide different sequence dataand contribute to distinct polymorphisms.That is because each semaphoront is the onlyexample of a particular combination of ossi-fied epipodials (characters 8 and 15) and othermature character states. A similar situation

occurs between semaphoronts S9 and S13,which also are mapped frequently to the samenode. However, S9 is never predicted to be thesame as S13’s other ‘‘twin,’’ S12, because S9and S12 have codings that clearly are distinctfrom one another (Table 1, Fig. 4).

Despite the wealth of information providedby OSA and its ability to incorporate missingdata during analysis, OSA may not always befeasible for paleontological data. A largesample size is necessary to obtain the frequen-cy support values that are used to identify themodal sequence. Additionally, the resolutionof the ontogenetic sequence is improved whenthe number of representative semaphoronts(OTUs) is greater than the number of events(characters), a situation that may not bepossible in studies of extinct taxa (Colbert1999). Moreover, because the method hasanalytical limitations similar to those affectingstandard cladistic analyses under parsimony,although OSA can handle missing data whenthere is enough clear ontogenetic informationto obtain a signal, when too much informationis missing sequences cannot be resolved, orthe fine resolution may be spurious. Forexample, OSA resulted in complete sequenceresolution for all the long bones in M. pelikani,even though the only specimens lacking anyossified long bones are missing data becauseof poor preservation. Finally, because of thesophistication and complexity of OSA, and thefact that all procedures currently are carriedout manually, the method is labor intensive.PAUP* can search a data set and output a fewhundred trees in a matter of seconds but eachtree must be examined one-by-one and themap is drawn by hand. Subsequently, allpredicted sequences need to be traced andtabulated and only at that point can statisticaland qualitative comparisons be made. Untilthe method is automated, large data sets andthose with a large proportion of missing datalimit the practicality of OSA.

If conservative approaches were taken, aconsensus of multiple methods could beutilized to formulate ontogenetic sequences.It is logical to combine size-based methodswith increasing number of ontogenetic events,as Schoch (2002) did for Apateon, although thistends to lead to many unresolved instances of


simultaneous appearance. Another possibilityis combining additional measures of maturity,such as the level of morphogenesis or sutureclosures (e.g., Brochu 1996; Colbert 1999), withsequence reconstruction, but thorough studiesof that nature are often not feasible oravailable for fossils. Additionally, the level ofmorphogenesis may not be consistent acrossan individual, which in vertebrates may berelated to dissociation between chondrifica-tion and ossification (Maisano 2002b; Maxwelland Larsson 2009).

Sequence Variation

Individuals that possess the same suite ofossified elements, regardless of size, are at thesame stage of maturity and should not beconsidered to exhibit polymorphisms. How-ever, because size-based methods rank indi-viduals by increasing length, larger, lessmature specimens will be encountered onlyafter the first appearance of elements in asmaller individual has been identified already.As a result, polymorphism in the form ofdelayed development (lack of an element) iseasily detectable, whereas accelerated devel-opment of an element will not be recognizedas polymorphic, because advanced individu-als will either be selected to represent firstappearances of elements or will be perceivedas delayed because ‘‘earlier’’ elements (inrelation to the structure of interest) remainundeveloped. Regarding the latter, individualsthat exhibit variation in the timing of devel-opment of multiple structures may actuallyindicate an early rather than delayed type ofpolymorphism because it is more parsimoni-ous to infer that only a single element isadvanced rather than multiple elements aredelayed, but the difference cannot be dis-cerned in practice (Harrison and Larsson2008).

Except under OSA, detection of polymor-phism is limited to retroactively inspecting thetotal sample of observed, fragmentary onto-genetic sequences for incongruent patterns offirst appearance. Because of that, the preva-lence of potential polymorphism detectedunder other methods likely is underestimatedrelative to the magnitude identified by OSA.Overall, strict polymorphisms (true ‘‘out-of-

order’’ sequences) were identified rarely,except under the majority-rule method andOSA (Table 2). Simultaneous polymorphismwas much more common and was identifiedfor both taxa under most of the methodsevaluated, although it was not detected whenusing the average OSA sequence for M.pelikani or for the trunk-length-based recon-struction for H. longicostatum.

The method based on increasing number ofontogenetic events can potentially mask strictpolymorphism because variations in the tim-ing of development will not appear ‘‘out-of-order.’’ Because only the number of ossifiedelements is taken into consideration, a moremature specimen that exhibits a delay in theossification of some bones (i.e., polymor-phism; based on a modal sequence) will tendto be inserted earlier in the hierarchy, equal tospecimens at an earlier stage of developmentthat also possess fewer elements. The suite ofontogenetic events present in those individu-als may be different, pointing to eithersimultaneous development or polymorphism.Therefore, reconstructions formulated fromincreasing number of ontogenetic events tendto limit strictly out-of-order cases of polymor-phism, but can greatly increase simultaneouspolymorphism from unresolved sequences.

In contrast, the majority-rule method yield-ed one of the lowest disparity estimates, butthat resulted mainly from a reduction in theamount of simultaneous polymorphism. Themajority-rule method inherently leads tosingleton developmental events in large sam-ple sizes because it is unlikely that elementswill be present in exactly the same number ofindividuals when large amounts of data aremissing. Methods based on length appear tobe less biased, although they exhibit fewerstrict polymorphisms relative to simultaneousones, suggesting a weaker ability to resolvethe timing of ontogenetic events.

One of the lowest disparity estimates wasprovided by the OSA results. OSA tends tolimit the number of strict polymorphismsidentified because the method returns a morefinely resolved sequence of ontogeneticevents, similar to the situation under themajority-rule method. Furthermore, it isknown from the frequency support values of


OSA that most of the non-semaphoront(redundant individuals) can be matched tomultiple ontogenetic sequences because of theprevalence of missing data. That observationimplies that individuals with large amounts ofmissing data also will support many of the 610predicted polymorphic sequences and thussuggests that the disparity estimate grosslyunderestimates the potential polymorphismpredicted under OSA. It remains uncertainhow many of the predicted polymorphismswould actually be observed in a real popula-tion and therefore whether disparity estimatesin general seriously underestimate true poly-morphism. An OSA study based on longitu-dinal growth series of human epipodialspredicted only one polymorphic sequence thatwas not represented in the sample (Colbert1999). However, that study did not involveoptimization of large amounts of missing data,which likely inflates the number of predictedsequences, and was essentially only mappingknown polymorphism, not inferring potentialsequences.

Differences in the amount and type ofpolymorphism detected under each methoddemonstrate that apparent polymorphismdepends on reconstruction methods and in-ferred ontogenetic sequences. However, ifparticular individuals are repeatedly identi-fied as expressing polymorphisms undermultiple reconstruction methods, it may beeasier to distinguish between apparent poly-morphism caused mainly by choice of meth-odology or differences in sequence resolution,and true polymorphism. Sequence polymor-phisms identified under multiple or all meth-ods of reconstruction, as opposed to onesdetected under only a single method, may bemore robust to methodological biases andthus are identified with a higher level ofconfidence as non-artifactual instances ofvariation.

Across all methods, polymorphisms morecommonly involved later-developing ele-ments (Tables 3, 4), a phenomenon predictedpreviously but not tested (Schoch 2010). Mostintraspecific variation in the ossification se-quence of M. pelikani involves the appearanceof the late-ossifying scapula, carpals, andtarsals. In fact, individuals CGH5, CGH2098,

and CGH727 were each identified by four ofthe methods of reconstruction as demonstrat-ing polymorphism for those elements, andCGH254 was identified by all methods (Sup-plementary Tables 1�3). From the separateossification sequences it is apparent that thescapula and epipodials ossify at nearly thesame time during development, and thusindividuals at the same stage, irrespective ofsize, may variably exhibit ossified epipodials,an ossified scapula, or both. The exact orderappears to be more labile than that of otherossification events, and in the case of carpaland tarsal polymorphism, may be related tothe dissociation of chondrification and ossifi-cation of epipodials in tetrapods. Studies ofsalamander limb morphology demonstratethat variation in adult epipodial patternresulting from condensation perturbations isa common phenomenon and support theobservation that the development of carpalsand tarsals is more labile than that of otherpostcranial elements (Shubin and Wake 2003).However, another explanation for the varia-tion observed in M. pelikani is that the scapulaand epipodials experience a higher degree ofpreservational bias relative to other postcrani-al elements. Both the scapula and epipodialsform late and may be more weakly ossifiedthan other bones during much of ontogeny,resulting in a more variable appearance in thefossil record.

Preservation and Sample Size

Developmental studies focused on fossilsoften must rely on low sample sizes, and evenwhen large numbers of individuals are avail-able, results may change when newly discov-ered specimens provide additional ontogeneticinformation. In studies of extinct taxa, speci-mens often are missing at both ends of the sizeextremes. Although the potential for such gapsmay be expected in studies based on fossils,missing data still can skew ideas about therelative timing of ossification events, particu-larly for early stages of development.

For example, lack of earlier developmentalstages can lead to a dense clustering ofdevelopmental events where structures seemto appear simultaneously. Low resolutionclearly is associated with gaps in growth


series; intermediate or earlier stages wouldhelp break up or spread out the distribution ofdevelopmental events into finer steps. Theeffect of those gaps is apparent in theontogenetic sequences reconstructed for H.longicostatum, which exhibit clustering and alow total number of ossification stages (Figs. 7,8). In M. pelikani, the trunk-length data setshows a similar pattern of few ossificationsteps, and the earliest step in the sequencecontains a large number of first appearances(Fig. 2). The skull-length reconstruction for thesame taxon, however, exhibits a first stagewith fewer total events, and overall there aremore developmental stages. That difference indistribution of events between the skull-lengthand trunk-length data sets is unexpectedconsidering that the samples share 35 speci-mens. Because of poor preservation thisproblem of event clustering affects analysesbased on fossils more than those based onextant taxa. That difference is clear when theearly ossification stages recovered for M.pelikani and H. longicostatum, as well thosereported for many temnospondyls (Schoch2003, 2004; Witzmann and Pfretzschner 2003;Witzmann 2006), are compared with the finerresolutions reported for living taxa (Rieppel1994; Smirnov and Vassilieva 2003; Banburyand Maglia 2006) (Figs. 9, 10).

The majority-rule method exhibits the op-posite effect. Clustering of ontogenetic eventsoccurs later in the sequence because elementsthat are present in only a few individuals aremore likely to share exactly the same numberof occurrences. That effect is exacerbated incases with very low sample sizes, like that ofH. longicostatum, where many elements arefound only in one or two specimens. Elementsthat occur earlier in the sequence, and thus arerepresented in more individuals, will tend tohave unique element counts because of differ-ences in preservation as well as separatetiming of appearance. Even if two elementsreally do develop simultaneously, preserva-tional bias in the fossil record makes itunlikely that the structures will be representedin exactly the same number of specimens.

Sample size also may have an effect on thedetection of polymorphism, at least when size-based methods are used. The comparatively

smaller amount of variation observed in thetrunk-length data set of M. pelikani and bothlength-based data sets of H. longicostatum maybe related to their smaller sample sizes.Natural variation may be more difficult todetect in smaller samples because largersamples, by including more individuals, willinherently capture more variation and thevariation may be more obvious. With largerdata sets a taphonomically altered specimen,or one expressing a rare polymorphism, is lesslikely to become the only evidence for thetiming of a particular ontogenetic event.

Comparison of Postcranial OssificationSequences

Comparisons between M. pelikani and H. long-icostatum.—Comparison of the skull-length-derived ossification sequences for M. pelikaniand H. longicostatum reveals many similaritiesin the development of the postcranial skeleton.Early in ontogeny, both exhibit ossification ofvertebrae, long bones, scales, and manydermal elements of the pectoral girdle (Fig.11). Although previously the centra werereported to ossify before the arches in H.longicostatum (Boisvert 2009), no evidence forthat pattern exists because even in the smallestindividuals known, arches and centra areossified already. The ilium also appearsrelatively early in both taxa. Later in ontogeny,the ischium ossifies, along with the haemalarches and sclerotic ring. In H. longicostatumossification of the pubis occurs at the sametime as the ischium, although in M. pelikani thepubis immediately follows the ischium, anobservation that partly may be the result ofthe larger sample size for M. pelikani, and thusbetter resolution. Finally, toward the end ofthe development of the postcranial skeleton,both taxa exhibit late ossification of theepipodials. No H. longicostatum used in mystudy has any ossified carpals, although largerindividuals with a fully ossified skeleton,including carpals, are known (Carroll andGaskill 1978).

A few key differences between the two taxainvolve the relative timing of appearance ofthe branchial plates, interclavicle, and scapula.In H. longicostatum ossification of the intercla-vicle occurs late relative to M. pelikani, after


FIGURE 9. Relative timing of ossification events in various temnospondyls and M. pelikani. Elements within the same boxappear at the same time; therefore, the order in which they are listed inside each box does not matter. The thick barsdenote relatively conserved events. Ossification sequences and modified skull images for Apateon from Schoch (2004),Sclerocephalus from Schoch (2003), Archegosaurus from Witzmann (2006), Micromelerpeton from Witzmann andPfretzschner (2003).


the appearance of all other elements except for

the tarsals. That delay in ossification was not

anticipated, because previous studies of Pa-

leozoic tetrapods suggested that dermal ele-

ments of the pectoral girdle ossify earlier than

endochondral components (e.g., Schoch 2003,

2004; Witzmann and Pfretzschner 2003; Witz-

mann 2006). In M. pelikani ossification of the

FIGURE 10. Relative timing of ossification events in Sclerocephalus, M. pelikani, and extant tetrapods. Details as in Figure9. Ossification sequence for Spea from Banbury and Maglia (2006), Lacerta from Rieppel (1994), Sclerocephalus fromSchoch (2003), Triturus from Smirnov and Vassilieva (2003). Sclerocephalus skull image modified from Schoch (2003).


scapula is particularly delayed relative to H.longicostatum, occurring near the end of theossification sequence. Branchial plates ossifyduring the earliest growth stage known for H.longicostatum, but they do not appear defini-tively in M. pelikani until about the time ofossification of the sclerotic ring, haemalarches, and ischium. Additionally, underOSA, the ‘‘increasing number of ontogeneticevents’’ method, and the majority-rule meth-od, in M. pelikani the long bones of theforelimb are ossified before those of the

hindlimb. However, in H. longicostatum ossi-fied elements of the hindlimb usually appeararound the same time as those of the forelimb,although under the majority-rule method forH. longicostatum, all hindlimb long bones areestimated to ossify before the forelimb ele-ments, a departure from the pattern found inmost tetrapods, with the exception of somefrogs (Maglia and Pugener 1998). Note again,however, that resolution of long bone ossifi-cation in both taxa may be an artifact ofmissing data, making it difficult to drawconclusions from such comparisons.

Comparison with Other Tetrapods.—Ossifica-tion sequence data based on skull length areavailable for many extinct temnospondyls.When data for M. pelikani are plotted on thesame relative time charts as other taxa, it isapparent that much of tetrapod postcranialdevelopment is conserved. However, M. pel-ikani exhibits some notable differences intiming when compared with the highlyconservative temnospondyls (Fig. 9). As inother lepospondyls (Carroll et al. 1999),ossification of the centra in M. pelikani occursat the earliest stage of development. Ossifica-tion of the sclerotic ring and the first appear-ance of the interclavicle both occur relativelylater, which resembles the shift in Apateon(Schoch 2004). Additionally, M. pelikani showsearly ossification of the pubis, before thecarpals and tarsals, and relatively late ossifi-cation of the scapula, close to the end ofpostcranial development.

When postcranial ossification in M. pelikaniis further compared with the sequence ofextant tetrapods and of temnospondyls to-gether, M. pelikani still exhibits early ossifica-tion of the pubis, although the pattern isshared with Lacerta (Fig. 10). In frogs andsalamanders, either the pubis never ossifies orit ossifies late in life, a pattern that stronglyresembles that of temnospondyls. The rela-tively late ossification of the scapula in M.pelikani stands out as unique. In general,salamanders do not completely ossify thepectoral girdle, but this usually results froma total loss of ossification events, rather thanjust a delay of endochondral ossification as isobserved in M. pelikani. In the latter, thedermal shoulder elements retain the ancestral

FIGURE 11. Relative timing of ossification events in M.pelikani and H. longicostatum, ordered according to skulllength. Details as in Figure 9.


pattern of early appearance and, although thescapula ossifies late, along with the epipo-dials, it achieves a high degree of ossificationin large individuals.

Microbrachis pelikani often is described aslimb-reduced because its forelimbs are smallrelative to its hindlimbs. That size disparitysuggests that M. pelikani was a hindlimb ortail-propelled swimmer, an idea that is sup-ported by the relatively early ossification ofthe pubis and late appearance of the scapulacorroborated by all methods of sequencereconstruction in my study, and by observa-tions of tail depth made by Milner (2008).Hyloplesion longicostatum, which also has rela-tively small forelimbs, exhibits approximatelythe same developmental pattern of the pubisossifying before epipodials and the scapulaeither near or after the ischium. The two taxamay be closely related (Carroll and Gaskill1978; Ruta et al. 2003; Ruta and Coates 2007),and the ossification sequence observed in bothcould be a synapomorphy related to function-al specializations. However, strong develop-ment of the hindlimbs might also result from abroader, ancestral pattern of lepospondyldevelopment that is coincident with forelimbreduction in the clade containing H. long-icostatum and M. pelikani. Previous work onthe postcranial development of paleognathousbirds suggested that delayed endochondralossification results from morphological reduc-tion in bone size (Maxwell and Larsson 2009).In M. pelikani the scapula does ossify late, butit is not reduced in size as much as the longbones and digits of the forearm, which aresome of the earliest elements to ossify.

Although there is scant information onossification and growth in extinct lepospondyls,study of the postcranial ossification sequence inM. pelikani and H. longicostatum makes itpossible to reevaluate previous observationsand hypotheses about development in thatclade. I am aware of only one other study inwhich a juvenile ‘‘microsaur’’ (Utaherpeton) waspositively identified and compared with anadult from the same taxon (Carroll and Chorn1995). Although that limited comparison cannotestablish a complete ossification sequence,information about the presence or absence ofelements in the Utaherpeton juvenile can be

compared with the ossification sequences of M.pelikani and H. longicostatum.

The Utaherpeton juvenile has ossified scales,long bones, neural arches, haemal arches, anilium, scapula, interclavicle, and clavicle (Car-roll and Chorn 1995). The neural-arch suture isstill present and although the pubis is unos-sified in the juvenile, it is present in an adult.That information provides a sequence ofdevelopment that differs from what is ob-served in M. pelikani and H. longicostatum. InUtaherpeton the scapula apparently is ossifiedbefore the ischium, pubis, and epipodials.That pattern is reminiscent of temnospondylsand is interesting because, in addition to beingone of the stratigraphically oldest lepospond-yls, Utaherpeton is basal to Microbrachis,Hyloplesion, and most other lepospondyls inmany phylogenetic analyses (Anderson 2001;Ruta et al. 2003; Anderson et al. 2008). Thatsuggests that the ancestral pattern of ossifica-tion in lepospondyls may be even more in linewith that of other tetrapods and that the lateossification of the scapula is a synapomorphyof a more derived lepospondyl group. Thetiming of ossification of the pubis cannot beevaluated with the data available, and thus itcannot be determined if that event is a generallepospondyl feature or not.

Conclusions

Differences among the reconstructions pro-vided by the alternative methods usuallyrepresented different degrees of sequenceresolution rather than major departures ofthe order in which morphological structuresossify. In light of the general agreementamong the different methods of ontogeneticsequence reconstruction, for extinct taxa,traditional reconstructions based on skulllength are justified when sample size is smallor poor preservation is a major factor. OSA isan equally objective method that has theadded benefit of being independent from size,age, or stage. When sample size is adequateand data allow for implementation, OSAshould be used because it provides bettersequence resolution and the ability to morefully explore observed and potential polymor-phism. All methods are affected by preserva-tional bias, and decisions must be made about


whether missing information results frombiology or taphonomy. The majority-rulemethod also is objective and perhaps simplerto use, but has greater potential to be affectedby preservational bias. However, the tech-nique may be useful in combination withlength-based methods because it can providemeasurements of preservational bias for indi-vidual elements.

Regardless of which method is used, andwhether the study relies on paleontological orneontological data, the amount and type ofvariation should be identified and discussed sothat future work is based on a solid foundationand does not rely solely on an isolated,chronological list of ‘‘typological’’ developmen-tal events. Individual variation is present in allpopulations, and thus, no matter which methodis used to reconstruct the ontogenetic sequence,only a hypothesis of the modal or averagesequence for the group is ever obtained. Withany reconstruction new information from newspecimens might modify previously recon-structed sequences, a fact not always remem-bered or welcomed by researchers.

Along with other lepospondyls, ‘‘micro-saurs’’ were considered to be direct developers(Baird 1965), and thus were expected to followa mode of development different from, andperhaps less complex and phylogeneticallyinformative than, that of temnospondyls.Although lepospondyls do appear to lack alarval stage and exhibit complete ossificationof the skull earlier than do many temnospond-yls, postcranial development does not differsignificantly from that of other Paleozoictetrapods. Instead, postcranial ossificationgenerally is conserved among extinct andextant tetrapods, and therefore the sequencesobserved for M. pelikani and for H. long-icostatum, with the exception of the lateossification of the scapula and early ossifica-tion of the pubis, follow an ancestral pattern.

Acknowledgments

This work resulted from a dissertationproduced at the University of Texas at Austin.I thank my advisor, C. J. Bell, and committeemembers, J. S. Anderson, D. Cannatella, T.Rowe, and J. Sprinkle, for advice and discus-sion regarding this paper. I thank K. Claeson

and E. Ekdale for reading early versions of thismanuscript, and B. Ekrt (NMP), J. Muller(MB), U. Gohlich (NHMW), S. Walsh (RSM),and C. Mehling (AMNH) for specimen access.I especially thank M. Colbert for assistancewith OSA. H. Larsson and an anonymousreviewer greatly improved the manuscript.Financial support for this portion of mydissertation was provided by the JacksonSchool of Geosciences and the Banks Fellow-ship (University of Texas at Austin), theSociety of Vertebrate Paleontology (EstesMemorial Grant), and the PaleontologicalSociety (Lane Student Award).

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Documents

Ontogenetic sequence reconstruction and sequence polymorphism in extinct taxa: an example using early tetrapods (Tetrapoda: Lepospondyli)