Skeletal heterochrony is associated with the anatomical specializations of snakes among squamate...

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Skeletal heterochrony is associated with the anatomical specializations of snakes among

squamate reptiles

Ingmar Werneburg1,2,*

, Marcelo R. Sánchez-Villagra1

1 Paläontologisches Institut und Museum der Universität Zürich, Karl-Schmid-Strasse 4,

8006 Zürich, Switzerland

2 Museum für Naturkunde, Leibniz-Institut für Evolutions- & Biodiversitätsforschung, an der

Humboldt-Universität zu Berlin, Invalidenstraße 43, 10115 Berlin / Germany

* [email protected]

ABSTRACT

Snakes possess a derived anatomy, characterized by limb reduction and reorganization of the

skull and internal organs. In order to understand the origin of snakes from an ontogenetic

point of view, we conducted comprehensive investigations on the timing of skeletal elements,

based on published and new data, and reconstructed the evolution of the ossification sequence

among squamates. We included for the first time Varanus, a critical taxon in phylogenetic

context. There is comprehensive delay in the onset of ossification of most skeletal elements

in snakes when compared to reference developmental events through evolution. We

hypothesize that progressing deceleration accompanied limb reduction and reorganization of

the snake skull. Molecular and morphological studies have suggested close relationship of

snakes to either amphisbaenians, scincids, geckos, iguanids, or varanids. Likewise, alternative

hypotheses on habitat for stem snakes have been postulated. Our comprehensive


heterochrony analyses detected developmental shifts in ossification for each hypothesis of

snake origin. Moreover, we show that reconstruction of ancestral developmental sequences is

a valuable tool to understand ontogenetic mechanisms associated with major evolutionary

changes and test homology hypotheses. The ‘supratemporal’ of snakes could be homolog to

squamosal of other squamates, which starts ossification early to become relatively large in

snakes.

Keywords

Skeletogenesis, fossils, embryos, Square Change Parsimony, Serpentes, Varanus

INTRODUCTION

Snakes are a diverse group of sauropsids, with more than 3,300 living species (Pincheira-

Donoso et al. 2013). When compared to other vertebrates, they exhibit numerous derived

anatomical features in their organ systems (e.g., they only have one lung) and, related to

those, derived physiological functions (Lüdicke 1964; Cundall and Irish 2008; Lawing et al.

2012). The body of snakes is associated with a multiplication of prelumbar vertebrae (body

elongation), and the reduction of limb and girdle elements (Figure 1D) (Apesteguía and Zaher

2006; Head and Polly 2007; Müller et al 2010). Paleontological data document some of the

transformations that have led to those specializations (Caldwell and Lee 1997; Longrich et al.

2009; Zaher et al. 2009). The Late Cretaceous species †Pachyrhachis problematicus, for

example, had reduced forelimbs, whereas the hind limbs were still well developed (Caldwell

and Lee 1997). Also, different degrees of pelvic girdle reduction are documented, including

that of the Late Cretaceous †Najash rionegrina and extant boine snakes (Figure 1E-G) (Zaher

et al. 2009).


The snake skull has a tube-like shape; several dermal bones are involved structurally in that

compact tube, whereas others are reduced (Mickoleit 2004). Many skull features functionally

relate to the feeding mode of eating large prey such as small land vertebrates, fishes, or eggs.

Snakes evolved highly mobile upper and lower jaws, which are connected via expandable

ligaments, and massive jaw musculature (Zaher 1994). Some snakes exhibit fangs, which,

together with large poison glands have important impact on the arrangement of skull bones

(Vonk et al. 2008).

The evolutionary transitions associated with most anatomical specializations of snakes are

still poorly understood, in part because their phylogenetic origin is highly debated (Conrad

2008; Hedges and Vidal 2009). Under one of the three major hypotheses on snake origins,

limb reduction and a ‘simplified’ and compact skull are proposed to be ancestral for snakes

(Gauthier et al. 2012). Snakes would be derived from fossorial ancestors, which would have

been adapted to unimpeded movement in subterraneous environment. This hypothesis

suggests a sistergroup relationship of snakes to the fossorial Amphisbaenia (“worm lizards”;

Figure 2A). A recent comprehensive morphological/molecular analysis incorporating critical

fossils, however, suggested a phylogenetic grouping of amphisbaenians with lacertid lizards

while advocating the independent evolution of body elongation and limblessness in

amphisbaenians and snakes and presented evidence for the dissociation in phylogeny of head

and postcranial specializations for fossoriality in squamate phylogeny [(Müller et al. 2011);

see Figure S1N].

Other morphological data and a total evidence analysis (Lee 2005) supported a position of

snakes within marine anguimorphs [but see (Müller et al. 2011)], which form the extinct

sister group to varanid lizards (Figure 2B). Compactness of the skull would be correlated to a

streamlining the head underwater and limb loss with a stepwise improvement of undulated


swimming. Under this second hypothesis the macropredatory mosasaurs, with their flipper-

like adapted limbs and their streamlined skulls and bodies, would be closely related to snakes

within Anguimorpha (Figure 2B).

Third, molecular data in some other studies suggested an unresolved position of snakes in

close relationship to iguanids and anguimorphs (Vidal and Hedges 2005; Pyron et al. 2013)

(Figure 2C), and support a terrestrial origin of snakes. Limb loss could have been associated

with unimpeded movement in low and dense vegetation of steppe-like landscapes and skull

compactness with the adaptation to feeding on large and hard prey.

Although all those hypotheses suggest different historical contexts in which snakes evolved

their anatomical specialization, a detailed scenario on how limbs or skull bones were lost

through evolution is lacking.

In order to investigate the evolutionary origin of snakes in anatomical, phylogenetic and

environmental perspectives, to date only adult morphological and molecular data have been

analyzed in broad scale analyses. However, a third dimension, ontogenetic information, was

missing so far to tackle the problem of snake origin from a mechanistic view point. Whereas

paleontology and molecular systematics focus mainly on adult specimens to gather

phylogenetic information, embryonic development represents a comprehensive and manifold

source of valuable information. Two principle mechanisms describe evolutionary changes in

embryonic morphogenesis, namely heterochrony and heterotopy. Heterochronies concern the

developmental timing of characters and are hypothesized to have fundamental impact on

shaping an adult structure. In mutual dependence with those, spatial reorganizations take

place during development, so called heterotopies. Comparative embryology is used to

investigate heterochrony and heterotopy – focusing on the evolution of new morphotypes as

part of organismic ontogenetic research programs.


For heterochrony analyses, in the last two decades different methods to calculate the timing

of developmental events within a phylogenetic framework have been presented (e.g.,

Bininda-Emonds et al. 2002; Harrison and Larsson 2008; Maxwell and Harrison 2009). In

our opinion, two methods are most valuable to understand evolutionary transformations.

A) Continuous analysis: in which the timing of developmental characters are scaled from 0 to

1 to allow comparison between even distantly related species, independent from how old or

different the embryos are (Germain and Laurin 2009). The scaled developmental characters

can be mapped on a given topology using square change parsimony. B) Event-pairing: an

established approach in the study of developmental sequence evolution (Velhagen 1997,

Smith 1997). In brief, the timing of a character A is compared to the timing of a character B;

if event A occurs before event B, the event-pair is coded as 0; if A and B occur

simultaneously, they are coded as 1; if A occurs after B, they are coded as 2. Based on event-

pairing, other methods such as Parsimov or PGi were developed (Jeffery et al. 2005; Harrison

and Larsson 2008) with and without different advantages (Werneburg and Sánchez-Villagra

2011; Ziermann et al. in press). Using both approaches, continuous analysis and event-

pairing, phylogenetic analyses can be performed and ancestral transformations in the timing

of developmental characters can be reconstructed. As a rule of thumb, the earlier a character

occurs in the developmental sequence of a given taxon, the larger it becomes in the adults;

and the later a developmental character occurs the smaller it is in the mature condition (e.g.,

Mehnert 1897, 1898; Sánchez-Villagra et al. 2008; Maxwell and Larsson 2009). The

prominence and size of a developmental structure has impact on the growth and expansion of

other structures, which, through evolution, results in the formation of new morphotypes

(heterotopic effect).


Concomitant with the specializations of adults, the chronology of skeletal development in

snakes is expected to be derived; however, this had not previously been studied within a

detailed evolutionary framework and by using quantitative methods (Irish 1989). For this, a

synthesis of diverse literature and the examination of key taxa have been lacking.

In order to study the developmental transformations that led to the specializations of snakes,

we analyze comparative data on the order of ossification of bones throughout the skeleton.

We examine sequences differ among squamates, we investigate which elements have been

reordered and their significance to the context of competing hypotheses of snake origin. We

hypothesize that the reduction of adult bones in snakes has led to significant heterochronies in

skeletal development. Given the more marked reduction of the forelimb and its earlier

phylogenetic reduction, as documented by the fossil record (Tchernov et al. 2000), we expect

delays in the forelimb ossification to have occurred in phylogeny before those in the hind

limb.

METHODS

Species and timing data. We compiled from the literature a dataset on the prehatching onset

of ossification of 142 skeletal elements for 30 lepidosaur species, with Alligator

mississippiensis as the outgroup (Figure S2-5, Table S1-3). We added the first data on

skeletogenesis of Varanus panoptes, a member of a critical genus to test snake relationships

(Figure 3; lab collection of Palaeontological Institute and Museum Zürich, PIMUZ

lab#2010.IW157-162, 2012.IW1-9; in sum, 15 specimens documented with µCT-scan). This

comparative analysis serves to examine all major alternative hypotheses on the origin of

snakes (Figure S1).


Developmental data on “basal” snakes are not available and are difficult to collect. As such,

the apomorphic heterochronic traits of snakes detected in our analyses are based on nested

taxa. Nevertheless, as we focus on major evolutionary changes in the present study, i.e. the

loss of limbs and skull reduction, the associated developmental features can be safely

assumed to characterize snakes in general. Moreover, ossification sequences in amniotes have

been demonstrated to exhibit conserved patterns among major clades and taxon-specific

changes in the timing of development can be expected to be characteristic for the whole

group (e.g., (Maxwell and Larsson 2009; Sánchez-Villagra et al. 2009; Werneburg et al.

2009; Hautier et al. 2013; Polachowski and Werneburg 2013; Koyabu et al. 2014)). As such,

the taxonomic sampling of other squamate groups in this work is also sufficient.

Character analysis and codification. All types of characters used herein – continuous and

relative ranked characters (see below) – were mapped on alternative topologies using PAUP*

(Swofford 2003) and Mesquite (Maddison and Maddison 2011) (Table S4). For all PAUP*-

reconstructions, heuristic search was applied with 10,000 replications (Table S5).

Continuous characters were created using timing data (Table S2-3), which were transformed

to a scale from 0 to 1 with the highest rank number representing ”1” (Germain and Laurin

2009) . Based on those ranks, cladistic characters were created following Laurin and Germain

(2011). Values from 0 to 0.1 were coded as state “0”, values until 0.2 were coded as state “1”

and so on up to character state “9”. For all species, relative timing ranks were coded (Table

S2-3) and phylogenetic reconstructions were performed. For embryonic series for which the

number of days of incubation and total incubation time were known (embryonic age) (Table

S1), absolute continuous data were also coded, transformed to cladistics characters, and

analyzed separately (Table S5E; Figure S6F). Parsimony reconstructions with PAUP* were

performed using one matrix including all species, one matrix including only those species


that have more than four ranks, and one matrix with species of more than five developmental

ranks (Figure S6).

Reconstruction of ancestral ossification sequences. Continuous characters were plotted onto

three geologically time scaled topologies, each implying a different ecological origin of

snakes (Figure 2A-C, S2-4, Table S6). For the topology of Vidal and Hedges (2005), we also

used a molecular time scale (Figure S5, Table S7). For nodes of unknown age (i.e., lower

taxonomic level), branch lengths were treated as equal.

The reconstructed timing-scores of the skeletal elements in snakes were compared to those of

the node that snakes share with their potential sister taxon. The average of all differences was

calculated and compared among the three major topologies. The smaller the difference, the

more parsimonious is the total character change between those two nodes. This comparison,

however, has no meaning for global squamate phylogeny.

Event-pair-mapping and Parsimov. Event-pairing was performed following Velhagen

(1997). Event-pair reconstruction only detects whether one character occurred before or after

another character in a particular node of the phylogeny. Using a cracking procedure, the

Parsimov-method reconstructs in which direction the change occurred through evolution,

namely which character moved against which other character (Jeffery et al. 2005). Using tie-

included event-pairs (i.e, simultaneous character pairs, coded as “1”), Parsimov (Jeffery et al.

2005) was applied (Werneburg and Sánchez-Villagra 2009), using 14 alternative topologies

as framework (Figure S2, Table S4). All characters were treated as unordered and equally

weighted. Gaps were treated as “missing”. Using event-pair-data, phylogenetic

reconstructions were conducted including and excluding ties (Table S5A-B). The latter

approach was applied to test the influence of potential pseudoshifts that are introduced by

working at low rank-resolution; i.e. when having less stages it is unknown whether characters

that are found to occur at the same time are just an artifact, because a higher resolved


sequence could identify more developmental stages in a sequence (Koyabu et al. 2011;

Werneburg and Sánchez-Villagra 2011).

For continuous analysis and for event-pairing, a Templeton test was performed, which

compares different phylogenetic topologies and, by listing tree length, it shows which

topology most parsimoniously explains the distribution of given characters.

RESULTS

Reconstruction of ancestral sequences. The patterns of ancestral ossification sequences in

the three major topologies of lepidosaur interrelationships (Figure S7-11, Table S8) do not

differ substantially among topologies. The ancestral cranial ossification sequences for all

nodes from Sauria leading to Serpentes are illustrated for topology B (Figure 4).

A progressive delay in the onset of ossification is detected for most bones, including the

clavicle, interclavicle, humerus, ulna, radius, tibia, fibula, metatarsals I-IV, and ischium. Few

bones show an acceleration, these being the palatine, pubis, caudal and sacral neural arches.

Many bones of the postcranium retain their approximate relative timing of ossification,

including the phalanges and metacarpals (Figure S7-11).

Tree lengths. When mapping characters on different trees, tree length explains how

wellcharacters distribute within the tree. The shorter tree length is the better the characters are

explained by the given topology. In every tree the characters distribute differently and will

result in a different interpretation of evolutionary transformation. As such, an assessment of

character distribution is a valuable step to estimate the significance of the detected

heterochronic changes.

When the 130 out of 142 parsimony-informative continuous characters are mapped on time-

scaled tree topologies A, B, and C, tree length provided by Mesquite was shortest for the


fossil time-calibrated topology C (tree length = 0.48), and longer for topologies A (0.61), B

(0.6238), and for the molecular time calibrated topology C (0.52). Topology C is best

explained by those characters.

A Templeton test, which does not consider branch lengths when mapping characters on a

tree, was performed in PAUP* using the timing data with scores from 0 to 9 (Table S9) and

resulted in the best distribution of characters within topology G (Figure S2G). Among the

three major topologies discussed herein (Figure 2), topology B presents the most

parsimonious distribution of ossification data. The same results were found for a Templeton

test using event-pair characters excluding ties (Table S10). Tree length of character mapping

cannot be used as a measure for the best topology (Assis and Rieppel 2011), as it only

illustrates the optimized distribution of timing data on alternative phylogenetic frameworks.

Transition to snakes. We were interested in the specific transition towards snakes in the

above mentioned topologies and counted the average differences between the reconstructed

ancestral timing scores of snakes and the node shared with their potential sister taxon. The

smaller the number the less anatomical changes need to be assumed to reach the snake

morphotype. The calculation results in a difference of 0.117 for topology A and 0.019 for

topology B. For topology C, it is 0.031 (fossil) and 0.048 (molecular time scale). As such,

topology B [Varanidae + Serpentes sensu Lee (2005)] provides the most parsimonious

explanation for overall character change between those two nodes herein and represents the

most parsimonious transition towards the snake developmental program.

Event-pair-mapping and Parsimov. For each of the 13 tested topologies, the consensus of

the apomorphic event-pair-characters and heterochronic shifts, which characterize ‘Serpentes

+ its potential sister group’ as well as the taxon ‘Serpentes’ alone, are listed in Tables S11-12.

The consensus are calculated as the overlap of accelerated and decelerated character

optimizations.


Parsimony-reconstructions using PAUP*. When using timing data to reconstruct a

phylogeny, none of the reconstructed trees mirrors any prior squamate topology and only few

closely related species were correctly reconstructed (Figure S6C-F, Table S5). The exclusion

of taxa with less than five or six ranks also did not result in any improvement in tree

recovery.

DISCUSSION

We detected a comprehensive delay in the relative timing of ossification of several skeletal

elements with respect to the remaining elements through lepidosaur evolution. This tendency

is most pronounced in snakes, in which several cranial and postcranial elements appear

extremely late. We hypothesize that the progressive delay in limb ossification was coupled

with limb reduction in snakes to the point of complete loss; i.e., ossification of the elements

was delayed to the point of absence. The timing of the onset of ossification is related to the

size of a morphological structure in the adult (Mehnert 1897, 1898; Sánchez-Villagra et al.

2008; Maxwell and Larsson 2009). This is an example of ‘developmental penetrance’, in

which adaptive changes in the adult phenotype are associated with corresponding changes in

earlier stages development (Richardson 1999; Bickelmann et al. 2012).

As reported in this comprehensive study of snakes and in previous works on specific clades

of squamates (Hugi et al. 2012), skeletal heterochronies occurred in association with

morphological transformations multiple times during evolution (Caldwell 2003; Siler and

Brown 2011). We predict that molecular markers of skeletogenesis may reveal traces of

element loss as has been shown in the limbs of archosaurs (de Bakker et al. 2013). Squamates

offer a rich subject of investigation on molecular mechanisms of limb reduction (Cohn and


Tickle 1999), with potentially different mechanisms involved in different clades, as has been

shown in digit reduction in mammals (Cooper et al. 2014).

Our reconstruction of ancestral ossification suggests that limb reduction first started in the

pectoral girdle and forelimb, with delays in the ossification of the clavicle, interclavicle,

scapula, humerus, ulna, and radius. This pattern is mirrored in the fossil record, where the

forelimb is reduced before the hind limb (Tchernov et al. 2000) (Figure 1). Later in evolution,

reduction started in the hind limb and pelvic girdle, with delays in ossification of the ischium,

tibia, fibula, calcaneum, and metatarsals I-IV (Figure 3-4). The onset of the ossification of

remaining limb elements (e.g., carpals, tarsals) was either accelerated or relatively constant

through lepidosaur evolution and we hypothesize a consequential co-reduction of those

elements based on their progressing loss of function.

The following cranial elements (Figure 3-4) show comprehensive deceleration in the onset of

ossification in snakes: Angular, dentary, frontal, maxilla, nasal, and parasphenoid. The

palatine shows considerable acceleration. The timing of those elements appears to correspond

with apomorphic features of the adult skull of Serpentes [as summarized by (Mickoleit

2004)], including: 1. the loss of median symphyses between the dentaries, 2. the loss of a

sutured contact between maxilla and premaxilla, and 3. a loose association of the maxilla

with the skull. In all those cases the bones start their growth relatively late and do not reach

their typically expanded squamate condition. Deceleration of other bones may be associated

with the spatially restricted tube-like shape of the skull in adult snakes that serves to cushion

biting forces when sucking the prey. A balanced timing of bones such as the nasals and

frontals may enable better arrangement within the derived “skull tube”. Finally, the palatal


region contains slender bones including the elongated palatine, which may be associated with

an earlier ossification onset for that bone.

The supratemporal (sensu (Cundall and Irish 2008)), the last remaining bone of the temporal

skull roof in snakes, remains constant in the relative onset of its ossification. Compared to

other lepidosaurs, however, the bone has an above-average adult prominence in advanced

snakes and one would expect an earlier onset of ossification of the supratemporal, or a higher

rate of growth. On the other hand, a different identity of the snake supratemporal is an

alternative hypothesis, such as the tabular, as was hypothesized before (McDowell 2008).

Given a potential sister group relationship of snakes to the fossil marine lizard Adriosaurus

(Anguimorpha) (Lee and Caldwell 2000), the snake ‘supratemporal’ – by positional criteria –

could instead represent the squamosal of lizards. The ossification of the bone in question is

considerably accelerated when compared to the reconstructed evolutionary trend in the timing

of the squamosal among squamates (red arrow with “?” in Figure 4). Hence, following the

simplified approach of assuming a constant growth rate after onset of ossification, the

‘supratemporal’ of snakes (sensu (Cundall and Irish 2008; Evans 2008)) could be interpreted

as squamosal of other squamates, which starts ossification early to become that large bone

visible in snakes. In this regard, it is worth mentioning that (in addition to a smaller

supratemporal) the squamosal is still present in ‘basal’ snakes such as Anilius.

We reconstructed developmental timing transformations for three alternative hypotheses for

snake origin (Tables S9-12). The simple use of event-pairs as characters to reconstruct

phylogeny did not recover trees in accordance with current understanding of squamate

phylogeny, not surprising given similar findings using ontogenetic timing data (Germain and

Laurin 2009). But ontogenetic data can contain phylogenetic information (e.g., (Mabee 1996,


2000; Maisano 2002, Haas 2003)) and the reconstruction of whole ancestral ossification

sequences (Figure 4), as exemplified herein, permits identifying differences in the

developmental timing of several bones. A sister group relationship to (fossorial)

amphisbaenians (Figure 2A), for example, is characterized by an acceleration of the

exoccipital relative to the frontal, which could be relevant for a posthatching stiffening of the

occiput against the body during burrowing. Potential support for a varanid sister-group

relationship (Figure 2B) or a relationship to iguanids + anguimorphs (Figure 2C) is

characterized by a common, comprehensive reorganization of the feeding apparatus, as

several bones of the jaws and the jaw adductor chamber are involved. No developmental shift

of the postcranium was detected as a consensus character in any group. This reinforces the

trend of delayed ossification affecting several parts of the skeleton as modules and not

individual bones alone.

CONCLUSIONS

We can confirm our hypothesis that in snakes the reduction of adult bones is associated with

significant heterochronies in skeletal development. The earlier phylogenetic reduction of the

forelimb compared to the hind limb is mirrored in the diverging delay of ossification that we

detected in related limb bones within squamate evolution. Moreover, several skull bones are

delayed in ossification within squamate evolution, resulting in the loss of those bones in

snakes.


Acknowledgements

We thank Christoph P. E. Zollikofer (Zürich) for use of micro-CT facilities, Mark N.

Hutchinson (Adelaide) for the collection of embryos of Varanus panoptes, Katja M.

Polachowski (Zürich) for the documentation of its ossification sequence, and Ashley Latimer

for suggestions for improvements in the text. Erin E. Maxwell (Stuttgart) and Robert J. Asher

(Cambridge) are thanked for their support. We are also grateful to P. David Polly

(Bloomington), Johannes Müller (Berlin) and three anonymous reviewers for their valuable

suggestions to former versions of the manuscript. The study was funded by the SNF grant

31003A_149605 granted to MRS-V

References

Apesteguía, S. and H. Zaher. 2006. A Cretaceous terrestrial snake with robust hindlimbs and a

sacrum. Nature 440:1037-1040.

Assis, L. C. S. and O. Rieppel. 2011. Are monophyly and synapomorphy the same or different?

Revisiting the role of morphology in phylogenetics. Cladistics 27:94-102.

Bickelmann, C., C. Mitgutsch, M. K. Richardson, R. Jimenez, M. A. G. de Bakker, and M. R. Sanchez-

Villagra. 2012. Transcriptional heterochrony in talpid mole autopods. EvoDevo 3.

Bininda-Emonds, O.R.P., J. E. Jeffery, and Coates, and M. K. Richardson. From Haeckel to event-

pairing: the evolution of developmental sequences. Theory Biosci. 2002;121:297-320.

Caldwell, M. W. and M. S. Y. Lee. 1997. A snake with legs from the marine Cretaceous of the Middle

East. Nature 386:705-709.

Cohn, M.J. and C. Tickle. Developmental basis of limblessness and axial patterning in snakes. Nature.

1999 3.6.1999;399:474-9.


Conrad, J. L. 2008. Phylogeny and systematics of squamata (Reptilia) based on morphology. Bulletin

of the American Museum of Natural History 310:182pp., 161 figs, 181 table.

Cooper, K., K. E. Sears, A. Uygur, J. Maier, K. Stephan-Backowski, M. Brosnahan, D. Antczak, J.

Skidmore, and C. Tabin. 2014. Patterning and post-patterning modes of evolutionary digit

loss in mammals. Nature 511.

Cundall, D. and F. Irish. 2008. The Snake Skull. Pp. 349-692 in C. Gans, A. S. Gaunt, K. Adler, A. S.

Gaunt, and K. Adler, eds. Morphology H. The Skull of the Lepidosauria. Society for the Study

of Amphibians and Reptiles, Salt Lake City.

de Bakker, M. A. G., D. A. Fowler, K. d. Oude, E. M. Dondorp, M. C. G. Navas, J. O. Horbanczuk, J.-Y.

Sire, D. Szczerbińska, and M. K. Richardson. 2013. Digit loss in archosaur evolution and the

interplay between selection and constraints. Nature 500:445-448.

Evans, S. E. 2008. The Skull of Lizards and Tuatara. Pp. 1-347 in C. Gans, A. S. Gaunt, and K. Adler,

eds. Morphology H. The Skull of the Lepidosauria. Society for the Study of Amphibians and

Reptiles, Salt Lake City.

Gauthier, J. A., M. Kearney, J. A. Maisano, O. Rieppel, and A. D. B. Behlke. 2012. Assembling the

squamate tree of life: perspectives from the phenotype and the fossil record. Bulletin of the

Peabody Museum of Natural History 53:3-308.

Germain, D. and M. Laurin. 2009. Evolution of ossification sequences in salamanders and urodele

origins assessed through event-pairing and new methods. Evolution & Development 11:170-

190.

Haas, A. 2003. Phylogeny of frogs as inferred from primarily larval characters (Amphibia: Anura).

Cladistics 19:23-89.

Harrison L, and H. Larsson. Estimating evolution of temporal sequence changes: a practical approach

to inferring ancestral developmental sequences and sequence heterochrony. Systematic

Biology. 2008;57(3):378-87.


Hautier, L., N. C. Bennett, H. Viljoen, L. Howard, M. C. Milinkovitch, A. C. Tzika, A. Goswami, and R. J.

Asher. 2013. Patterns of ossification in southern versus northern placental mammals.

Evolution 67:1994-2010.

Head, J. J. and P. D. Polly. 2007. Dissociation of somatic growth from segmentation drives gigantism

in snakes. Biology Letters:1-3 [doi:10.1098/rsbl.2007.0069].

Hedges, S. B. and N. Vidal. 2009. Lizards, snakes, and amphisbaenians. Pp. 383-389 in S. B. Hedges,

and S. Kumar, eds. The TimeTree of Life. Oxford University Press, New York.

Hugi, J., M. N. Hutchinson, D. Koyabu, and M. R. Sanchez-Villagra. 2012. Heterochronic shifts in the

ossification sequences of surface- and subsurface-dwelling skinks are correlated with the

degree of limb reduction. Zoology 115:188-198.

Irish, F. J. 1989. The role of heterochrony in the origin of a novel bauplan: evolution of the ophidian

skull. Geobios 22:227-233.

Jeffery, J. E., O. R. P. Bininda-Emonds, M. I. Coates, and M. K. Richardson. 2005. A new technique for

identifying sequence heterochrony. Systematic Biology 54:230-240.

Koyabu, D., H. Endo, C. Mitgutsch, G. Suwa, K. C. Catania, C. P. E. Zollikofer, S. Oda, K. Koyasu, M.

Ando, and M. R. Sánchez-Villagra. 2011. Heterochrony and developmental modularity of

cranial osteogenesis in lipotyphlan mammals. Evo Devo (BMC) 2.

Koyabu, D., I. Werneburg, N. Morimoto, C. P. E. Zollikofer, A. M. Forasiepi, H. Endo, J. Kimura, S. D.

Ohdachi, S. N. Truong, and M. R. Sánchez-Villagra. 2014. Mammalian skull heterochrony

reveals modular evolution and a link between cranial development and brain size. Nature

Communications 5:3625 (3628 pages).

Laurin, M. and D. Germain. 2011. Developmental characters in phylogenetic inference, and their

absolute timing information. Systematic Biology 60:630-644.

Lawing, A. M., J. J. Head, and P. D. Polly. 2012. The ecology of morphology: the ecometrics of

locomotion and macroenvironment in North American snakes, in Pp. 117-146 in J. Louys, ed.

Palaeontology in Ecology and Conservation. Springer, New York.


Lee, M. S. Y. 2005. Molecular evidence and marine snake origins. Biology Letters 1:227-230.

Lee, M. S. Y. and M. W. Caldwell. 2000. Adriosaurus and the affinities of mosasaurs, dolichosaurs,

and snakes. Journal of Paleontology 74:915-937.

Longrich, N. R., B.-A. S. Bhullar, and J. A. Gauthier. 2009. A transitional snake from the Late

Cretaceous period of North America. Nature 488:205-208.

Lüdicke, M. 1964. andbuch der oologie and . l te 1, Sauropsida, Allgemeines, Reptilia.

Lieferung 5-6, Ordnung der Klasse Reptilia, Serpentes. W. de Gruyter, Berlin.

Mabee, P. M. 1996. Reassessing the ontogenetic criterion: a response to Patterson. Cladistics

12:169-176.

Mabee, P. M. 2000. The usefulnes of ontogeny in morphological characters in J. J. Wiens, ed.

Phylogenetic Analysis of Morphological Data. Smithonian Institution Press, Washington,

London.

Maisano, J. A. 2002. The potential utility of postnatal skeletal developmental patterns in squamate

phylogenetics. Zoological Journal of the Linnean Society 136:277-313.

Maddison, W. P. and D. R. Maddison. 2011. Mesquite: a modular system for evolutionary analysis.

Version 2.75, http://mesquiteproject.org.

Maxwell, E. E. and H. C. E. Larsson. 2009. Comparative ossification sequence and skeletal

development of the postcranium of palaeognathous birds (Aves: Palaeognathae). Zoological

Journal of the Linnean Society 157:169-196.

Maxwell, E. E. and L. B. Harrison 2009. Methods for the analysis of developmental sequence data.

Evolution & Development. 11(1):109-19.

McDowell, S. B. 2008. The Skull of Serpentes. Pp. 467-620 in C. Gans, A. S. Gaunt, K. Adler, A. S.

Gaunt, and K. Adler, eds. Morphology I. The Skull and Appendicular Locomotor Apparatus of

Lepidosauria. Society for the Study of Amphibians and Reptiles, Salt Lake City.

Mehnert, E. 1897. Kainogenesis als Ausdruck differenter phylogenetischer Energien. Verlag von

Gustav Fischer, Jena.


Mehnert, E. 1898. Biomechanik erschlossen aus dem Principe der Organogenese. Gustav Fischer,

Jena.

Mickoleit, G. 2004. Phylogenetische Systematik der Wirbeltiere. Verlag Dr. Friedrich Pfeil, München.

Müller, J., T. M. Scheyer, J. J. Head, P. M. Barrett, I. Werneburg, P. G. P. Ericson, D. Pol, and M. R.

Sánchez-Villagra 2010. Homeotic effects, somitogenesis and the evolution of vertebral

numbers in recent and fossil amniotes. Proceedings of the National Academy, USA.

107:2118-23.

Müller, J., C. A. Hipsley, J. J. Head, N. Kardjilov, A. Hilger, M. Wuttke, and R. R. Reisz. 2011. Eocene

lizard from Germany reveals amphisbaenian origins. Nature 473:364-367.

Pincheira-Doroso, D., A.M. Bauer, S. Meiri, and P. Uetz 2013. Global taxonomic diversity of living

reptiles. PLOS One. 8(3):e59741.

Polachowski, K. M. and I. Werneburg. 2013. Late embryos and bony skull development in

Bothropoides jararaca (Serpentes, Viperidae). Zoology 116:36-63.

Pyron, R. A., F. T. Burbrink, and J. J. Wiens. 2013. A phylogeny and revised classification of Squamata,

including 4161 species of lizards and snakes. BMC Evolutionary Biology 13.

Richardson, M. K. 1999. Vertebrate evolution: the developmental origins of adult variation.

BioEssays 21:604–613.

Sánchez-Villagra, M. R., A. Goswami, V. Weisbecker, O. Mock, and S. Kuratani. 2008. Conserved

relative timing of cranial ossification patterns in early mammalian evolution. Evolution &

Development 10:519-530.

Sánchez-Villagra, M. R., H. Müller, C. A. Sheil, T. M. Scheyer, H. Nagashima, and S. Kuratani. 2009.

Skeletal development in the Chinese soft-shelled turtle Pelodiscus sinensis (Testudines:

Trionychidae). Journal of Morphology 270:1381-1399.


Smith, K. K. Comparative patterns of craniofacial development in eutherian and metatherian

mammals. Evolution. 1997;51(5):1663-78.

Swofford, D. L. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). 4 ed.

Sunderland, Massachusetts: Sinauer Associates.

Tchernov, E., O. Rieppel, H. Zaher, M. J. Polcyn, and L. L. Jacobs. 2000. A fossil snake with limbs.

Science 287:2010-2012.

Velhagen, W. A., Jr. 1997. Analyzing developmental sequences using sequence units. Systematic

Biology 46:204-210.

Vidal, N. and S. B. Hedges. 2005. The phylogeny of squamate reptiles (lizards, snakes, and

amphisbaenians) inferred from nine nuclear protein-coding genes. Comptes Rendus

Biologies 328:1000-1008.

Vonk, F.J., J. F. Admiraal, K. Jackson, R. Reshef, M. A. G. de Bakker, K. Vanderschoot, I. van den Berge,

M. van Atten, E. Burgerhout, A. Beck, P. J. Mirtschin, E. Kochva, F. Witte, B. G. Fry, A. E.

Woods, and M. K. Richardson. 2008. Evolutionary origin and development of snake fangs.

Nature 454, 630-633

Werneburg, I., J. Hugi, J. Müller, and M. R. Sánchez-Villagra. 2009. Embryogenesis and ossification of

Emydura subglobosa (Testudines, Pleurodira, Chelidae) and patterns of turtle development.

Developmental Dynamics 238:2770-2786, doi: 2710.1002/dvdy.22104, two Supplements.

Werneburg, I. and M. R. Sánchez-Villagra. 2009. Timing of organogenesis support basal position of

turtles in the amniote tree of life. BMC Evolutionary Biology 9.

Werneburg, I. and M. R. Sánchez-Villagra. 2011. The early development of the echidna, Tachyglossus

aculeatus (Mammalia: Monotremata), and patterns of mammalian development. Acta

Zoologica 82:75-88.

Wiens, J. J., C. A. Kuczynski, T. Townsend, T. W. Reeder, D. G. Mulcahy, and J. W. Sites. 2010.

Combining phylogenomics and fossils in higher-level squamate reptile phylogeny: molecular

data change the placement of fossil taxa. Systematic Biology 59:674-688.


Zaher, H. 1994. Comments on the evolution of the jaw adductor musculature of snakes. Zoological

Journal of the Linnean Society 111:339-384.

Zaher, H., S. Apesteguía, and C. A. Scanferla. 2009. The anatomy of the upper Cretaceous snake

Najash rionegrina Apesteguía & Zaher, 2006, and the evolution of limblessness in snakes.

Zoological Journal of the Linnean Society 156:801-826.

Ziermann J.M., C. Mitgutsch, and L. Olsson. Analyzing developmental sequences with Parsimov - a

case study of cranial muscle development in anuran larvae. In press. Journal of Experimental

Zoology Part B: Molecular and Developmental Evolution. doi: 10.1002/jez.b.22566.


Figure 1. Skull and limb evolution. A-C) Skull reconstructions of A) Heloderma

(Varanomorpha), B) †Coniophis (a potential sister taxon to Ophidia), and the C) extant

macrostomatan snake Epicrates; modified from Longrich et al. (2009). D) Skeleton of

†Pachyrhachis problematicus with reduced forelimbs but present hind limbs; modified from

Caldwell and Lee (1997). D-F) Different degrees of hind limb and pelvic girdle reduction

among snakes, including D) †Najash rionegrina, E) †P. problematicus, and F) a boine snake;

modified from Zaher et al. (2009).


Figure 2. Three major hypotheses on the phylogenetic position of snakes (Serpentes). The

inferred ecological origin of snakes is A) fossorial (Gauthier et al. 2012), B) aquatic (Lee

2005), or C) fully land adapted (Vidal and Hedges 2005; Wiens et al. 2010; Pyron et al.

2013) environment. For further alternative topologies see electronic supplementary material

(Figure S1). In the study of Lee (2005) (B), snakes are situated within a paraphyletic clade of

fossil marine anguimorphs to which the illustrated mosasaurid belongs (but see (Müller et al.

2011): Figure S1N).


Figure 3. Skeletons of a late-stage snake and a late-stage varanid embryo (µCT-scan). A)

Skull and anterior vertebrae of the viper snake Bothropoides jararaca (PIMUZ lab#

2010.IW155). B) Skeleton of Varanus panoptes (PIMUZ lab#2010.IW162). Color code in V.

panoptes indicates the general direction in the change of ossification timing through

squamate evolution. Grey scale = relatively constant timing through squamate evolution,

orange = progressive delay, blue = progressive acceleration. Delay in the onset of ossification

is hypothesized to result in reduced adult bones. For further details see Figures 4, S7-11.


Figure 4. Reconstructed ancestral ossification sequences of selected skeletal elements using

continuous characters; exemplified for topology B (Figures 2B, S1B). For full labeling, all

142 bones, and reconstructions for topology A and C, see Figures S7-11. Arrows indicate

major directions in the change of ossification timing through squamate evolution. No arrows

are shown for bones with minute or strongly uncoordinated change in timing.

Skeletal heterochrony is associated with the anatomical specializations of snakes among squamate...

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