Embed Size (px)
Citation preview
LETTERdoi:10.1038/nature14042
Evolution of the snake body form reveals homoplasyin amniote Hox gene functionJason J. Head1 & P. David Polly2
Hox genes regulate regionalization of the axial skeleton in vertebrates1–7,and changes in their expression have been proposed to be a funda-mental mechanism driving the evolution of new body forms8–14. Theorigin of the snake-like body form, with its deregionalized pre-cloacalaxial skeleton, has been explained as either homogenization of Hoxgene expression domains9, or retention of standard vertebrate Hoxdomains with alteration of downstream expression that suppressesdevelopment of distinct regions10–13. Both models assume a highlyregionalized ancestor, but the extent of deregionalization of the pri-maxial domain (vertebrae, dorsal ribs) of the skeleton in snake-likebody forms has never been analysed. Here we combine geometricmorphometrics and maximum-likelihood analysis to show that thepre-cloacal primaxial domain of elongate, limb-reduced lizards andsnakes is not deregionalized compared with limbed taxa, and thatthe phylogenetic structure of primaxial morphology in reptiles doesnot support a loss of regionalization in the evolution of snakes. Wedemonstrate that morphometric regional boundaries correspond tomapped gene expression domains in snakes, suggesting that theirprimaxial domain is patterned by a normally functional Hox code.Comparison of primaxial osteology in fossil and modern amnioteswith Hox gene distributions within Amniota indicates that a func-tional, sequentially expressed Hox code patterned a subtle morpho-logical gradient along the anterior–posterior axis in stem membersof amniote clades and extant lizards, including snakes. The highlyregionalized skeletons of extant archosaurs and mammals result fromindependent evolution in the Hox code and do not represent ancestralconditions for clades with snake-like body forms. The developmentalorigin of snakes is best explained by decoupling of the primaxial andabaxial domains and by increases in somite number15, not by changesin the function of primaxial Hox genes9,10.
In Amniota (Mammalia 1 Reptilia), Hox genes are expressed sequen-tially in the somitic mesoderm, resulting in a series of distinct anato-mical regions along the anterior–posterior axis of the vertebral column.Anatomical boundaries coincide with anterior borders of Hox geneexpression or areas where expression of two genes overlaps4,5. In Squa-mata (lizards, including snakes), the pre-cloacal vertebral column is lessdifferentiated than in highly regionalized mammals and extant archo-saurs. Vertebrae possessing synapophyses that articulate with dorsal ribsextend from the first post-atlanto-axial vertebra to the sacrum in manysquamates16 (Fig. 1 and Extended Data Fig. 1a). In limbed lizards, tworegional boundaries, cervical–thoracic and thoracic–lumbar, are knownto correspond to Hox gene expression patterns10,12. These boundariesare not recognized in snakes and snake-like squamates, which are con-sidered to possess ‘deregionalized’11 axial skeletons with increased num-bers of vertebrae and ribs and reduction or loss of limbs and sternum16.
Two conflicting hypotheses have been proposed for the role of Hoxgenes in the evolution of the snake-like axial skeleton on the basis ofdomain mapping and transgenic expression9–13. In the first hypothesis,loss of regionalization is caused by upstream modification of Hox expres-sion and (or resulting in) a shift of HoxC6 and HoxC8 domains, which areassociated with the thoracic region in mammals and archosaurs, forward
to the first post-atlanto-axial vertebral position9. In the second, loss ofregionalization in snakes is caused not by shifts in the boundaries ofHox expression, but by downstream changes in cis-regulation10–13. Bothhypotheses invoke modifications to Hox activity in the paraxial meso-derm, which forms the primaxial skeleton (vertebrae, dorsal ribs17), butaxial regionalization is at least partially dependent upon spatial relation-ships with the abaxial skeleton (limbs, sternum17,18), which is derivedfrom lateral plate mesoderm and has independent Hox regulation4,5.The extent to which the primaxial domain has become homogenizedin clades with snake-like body forms has, to our knowledge, never beenexamined in a comparative phylogenetic context.
To test the hypothesis that the primaxial skeleton of snakes and snake-like squamates is deregionalized relative to limbed amniotes, we per-formed geometric morphometric analysis on vertebral morphology tomeasure quantitatively intracolumnar shape variance and combinedit with a maximum-likelihood estimation of the number of regionsand positions of regional boundaries in the pre-cloacal skeleton of rep-resentative taxa based on segmented linear regression (Methods, ExtendedData Fig. 2 and Extended Data Table 1). To capture the axial gradientin shape, we placed 12 homologous landmarks on vertebrae along thepre-cloacal skeleton19 (Fig. 1, Methods and Extended Data Table 2). Weincluded comparisons with Alligator mississippiensis and Mus musculusbecause Hox expression boundaries in their regionalized axial skele-tons are well documented2–6,20 (Methods and Extended Data Fig. 3).
We found that total intracolumnar shape variance in the primaxialdomain was substantially less in all squamates than in Alligator (Fig. 2a).Mean variance was significantly lower in snake-like squamates than in
1Department of Earth and Atmospheric Sciences and Nebraska State Museum of Natural History, University of Nebraska–Lincoln, Lincoln, Nebraska 68588-0340, USA. 2Departments of GeologicalSciences, Biology and Anthropology, Indiana University, Bloomington, Indiana 47405-1405, USA.
1
12 11
10
9
8
7
6
4
5
23
a
b
Figure 1 | Morphological variation in the pre-cloacal vertebral column oflimbed lizards and snakes. a, b, Pogona vitticeps (a) and Pantherophis guttatus(b) pre-cloacal vertebrae in anterior view, from left: first post-atlanto-axial,mid-trunk and posterior-most pre-cloacal vertebrae. Numbered landmarksshown on mid-trunk vertebra of Pogona were used to characterize vertebralshape (Extended Data Table 2).
G2015 Macmillan Publishers Limited. All rights reserved
8 6 | N A T U R E | V O L 5 2 0 | 2 A P R I L 2 0 1 5
limbed taxa (see Methods). Nevertheless, there was no consistent dif-ference in shape variance between limbed and snake-like squamates,and the range of variances in snake-like squamates exceeds the rangein limbed taxa (Fig. 2b). Several snake taxa have greater intracolumnarshape variance than any of the sampled limbed squamates, even afterstandardizing for differences in the number of vertebrae.
The number of primaxial regions in snakes and snake-like lizards doesnot systematically differ from limbed squamates and Alligator (Fig. 2c, d).Three or four regions were found in all taxa, irrespective of the presenceor absence of limbs or the total number of vertebrae. The origin of snake-like body forms was not associated with a reduction in the number ofregions when we tested four competing evolutionary models of dereg-ionalization (Fig. 2d, Methods and Extended Data Table 3). The modelin which limbed squamates and Alligator have four regions and thisis reduced to three in snake-like taxa was no better supported than thehypothesis that all squamates share three regions and Alligator has four(relative support 5 0.6 for both hypotheses). Models in which snake-liketaxa have two regions and limbed taxa have either three or four regions
had virtually no support (relative support 5 0). These results indicatethat, although average shape variance within and between individualprimaxial regions of snake-like taxa is less than in their limbed rela-tives, there was no reduction in the number of regions or changes inthe relative location of regional boundaries associated with the originof snakes or snake-like taxa.
To determine if morphometric regional boundaries are associatedwith Hox expression, we fit regional models to entire pre-cloacal pri-maxial skeletons of representative taxa and compared best-fit results tomapped Hox expression boundaries3,4,20. Four regions were found inmost taxa (Extended Data Fig. 4 and Extended Data Table 4). In Musand Alligator, four-region models recovered morphometric boundariesthat either exactly matched Hox expression boundaries for regional tran-sitions or were within one vertebral position of boundaries (Fig. 3 andSupplementary Information).
In the snake Pantherophis guttatus, the best-fit regional boundariescorrespond to Hox expression domains that govern the cervical–thoracictransition and the thoracic region in limbed amniotes (Fig. 3). The
Alligator mississippiensis
Cordylus giganteus
Eumeces schneideri
Corucia zebrata
Amphisbaeana albaTupinambis teguixin
Neusticurus rudis
Iguana iguana
Physignathus cocincinus
Pogona vitticeps
Pseudopus apodusHeloderma suspectum
Varanus bengalensis
Typhlops punctatusRena dulcisAnilius scytaleTropidophis canus Xenopeltis unicolorLoxocemus bicolorLeiopython albertisii Morelia spilota Python regius Charina bottaeLichanura trivirgataCandoia carinata Eryx johniiBoa constrictorCorallus caninusEunectes notaeusCalabaria reinhardtii Acrantophis dumerili Sanzinia madagascarensis Cylindrophis ruffus Uropeltis woodmasoniAcrochordus granulatusOxyrhabdium leporinumCrotalus ruberAgkistrodon contortixDaboia russelliCerberus rynchopsErpeton tentaculatumMadagascarophis colubrinaActractaspis irregularisHydrophis semperiMicrurus fulviusNaja nigricollis Heterodon platyrhinosWaglerophis merremiiThamnophis sirtalisBoiga irregularisPtyas mucosusColuber constrictor Pantherophis guttatus
1 10 20 30 40 50 60 70 80 90 100
0.02
Intracolumnar
shape variance
–260.02
–136.99
–154.00
–143.74
–154.00
–149.98
–136.99
–154.00
–154.00
–144.78
–154.00
–154.00
–154.00
–154.00
–154.00
–150.69
–154.00
–154.00
–154.00
–154.00
–154.00
–154.00
–154.00
–154.00
–154.00
–150.52
–154.00
–154.00
–154.00
–154.00
–154.00
–154.00
–154.00
–154.00
–154.00
–148.05
–146.09
–154.00
–154.00
–154.00
–154.00
–154.00
–134.34
–154.00
–154.00
–154.00
–134.62
–154.00
–154.00
–143.74
–154.00
–154.00
–154.00
AICc
4
4
4
4
4
4
4
4
4
3
4
3
3
2
3
3
3
3
3
3
3
4
3
4
3
2
Evolutionary models of
regionalization
RS
434
3
a
b
Snake-like0.002
0.006
0.01
0.014
0.018
Variance
Limbed
d
3
3
4
4
3
3
2
2
c
3
3
2
2
0.6 0.6 0.0 0.0Per cent body length 1.0
Figure 2 | Regional boundaries, evolutionary models of regional changes,and intracolumnar variance. a, Consensus phylogeny of selected taxa.Terminal branch lengths are scaled to intracolumnar shape variance. b, Boxplot of intracolumnar variances in limbed (n 5 10 specimens) and snake-like(n 5 42 specimens) squamates. c, Regional boundaries in primaxial domainsfor each taxon subsampled at 5% intervals. Coloured cells represent vertebrae in
different regions of the best-fit model, for which corrected Akaike informationcriterion (AICc) scores are given. Taxa in bold are snakes and snake-likesquamates. d, Best-fit distribution of regions (left) compared with four modelsfor evolutionary changes in regionalization. Each is depicted by the number ofregions (2 to 4) expected in limbed and snake-like taxa. RS, relative support(Methods).
LETTER RESEARCH
G2015 Macmillan Publishers Limited. All rights reserved
2 A P R I L 2 0 1 5 | V O L 5 2 0 | N A T U R E | 8 7
boundary between the first and second regions occurs between post-atlanto-axial vertebrae 10–11, which corresponds to the diffuse anteriorexpression boundary of HoxC6 (ref. 10). This correspondence was alsorecovered in limbed squamates (Supplementary Information). The boun-dary between the second and third morphological regions occurs betweenpost-axial vertebrae 49–50, which falls within the diffuse anterior expres-sion boundary of HoxC8 (ref. 10) and is only five vertebrae posteriorto the anterior expression region for HoxA7 (ref. 10). The boundarybetween the third and fourth morphological regions in Pantherophisoccurs between post-axial vertebrae 180–181, which is anterior to expres-sion of HoxA10 and C10 near somites 195 to 210 (refs 10, 12). Thisapparent discrepancy may arise from individual, possibly sex-linked21,differences in vertebral number between our sample and Hox-mappedspecimens (Supplementary Information). Regardless, the fit of the fourthmorphometric regional boundary to Hox10 expression boundaries wasstatistically indistinguishable from the best-fit model (Methods andExtended Data Fig. 5).
Regional transitions in Pantherophis and the other squamates in ourstudy are gradational, unlike the more abrupt boundaries of Mus orAlligator, in which differences in the presence, articulation or fusion ofvertebral processes and ribs add to regional differentiation (ExtendedData Fig. 1 and Fig. 4). Both boundary types emerge from Hox geneexpression. HoxC8 expression is associated with relative sizes of theneural arch and apophyses20, and the expression of this gene is gradedover a series of segments in snakes rather than having a sharply definedboundary as in limbed taxa10. Topographic correlation between Hoxexpression boundaries and morphometric regions in Pantherophis (Fig. 3)is evidence that Hox domains in the snake primaxial skeleton are func-tional, even though regional boundaries lack discrete structural changesin processes and ribs.
Paleozoic amniotes, including stem members of Reptilia and Mam-malia, possessed a comparatively homogeneous vertebral column anddorsal rib cage that lacked the distinct regional boundaries found in mam-mals and extant archosaurs, even though mapped domains for extantreptiles, mammals and anamniotes4,10,20,22,23 indicate that a fully regio-nalized and functional set of Hox genes along the anterior–posterioraxis was ancestral for Amniota (Fig. 4). Our evidence indicates that thespecific functions of Hox genes in patterning the regionalized primaxialdomain of mammals and extant archosaurs are probably homoplastic
Alligator mississippiensis
C3
C4
C5
C6
C7
C8
C9
T1
T2
T3
T4
T5
T6
T7
T8
T9
T1
0L1
L2
L3
L4
L5
HoxB4
HoxC6
HoxA7
HoxC8
HoxC4
HoxD4
HoxA5
HoxB5
Pantherophis guttatus
HoxA10
HoxC6
HoxC8
HoxA7
HoxB4
HoxC6
HoxA7
HoxC8
HoxC4
HoxD4
HoxA5
HoxB5
Mus musculus
Hox9Hox10
C3
C4
C5
C6
C7
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T13
L1
L2
L3
L4
L5
L6
T12
V3 V224
V10
V49
V181
Figure 3 | Correspondence between Hox expression domains andmorphometric boundaries for four-region models of primaxialregionalization. Coloured bars represent expression domains for Mus4,20
and Alligator20, and the range of anterior expression boundaries forPantherophis10–12. Hox expression domains for the thoracic–lumbar transitionin Alligator have not yet been mapped20. Cells represent individual vertebrae ineach region for the entire pre-cloacal/pre-sacral vertebral column in each taxon.Cell colours represent morphometric regions. Grey bars indicate regions ofoverlap between genes and morphometric regions. C, cervical; L, lumbar;T, thoracic; V, vertebra.
Seymouria†Captorhinus†
Alligator Uromastyx Lichanura
Thrinaxodon†
Mus
Cen.
Pal.
Anterior
Posterior
Hox45
67
89
10
1
2
3
Age
(Myr ago)
0
80
160
240
320
Meso
zo
ic
5
4
Figure 4 | Time-calibrated phylogeny of selected extant and fossil amniotes,illustrating pre-cloacal and pre-sacral primaxial skeletal regionalizationand the generalized ancestral amniote pattern of Hox expression. Nodenumbers label the total clades for Amniota (1), Reptilia (2) and Mammalia (3),and the crown clades for Reptilia (4) and Squamata (5). Archosauria is
represented by Alligator, crown Mammalia is represented by Mus. Colouredbars represent relative positions of anterior expression domain boundaries forHox4–10 paralogues along the anterior–posterior axis in Amniota. SeeSupplementary Information for data sources. Cen., Cenozoic; Pal., Palaeozoic.Daggers indicate fossil taxa.
RESEARCH LETTER
G2015 Macmillan Publishers Limited. All rights reserved
8 8 | N A T U R E | V O L 5 2 0 | 2 A P R I L 2 0 1 5
exaptations of an ancestral Hox code whose original function in amni-otes was regulation of subtle gradations in primaxial morphology. Thispattern is conserved in snakes and most other squamates (Fig. 4).
We conclude that the origin of snakes was not associated with dere-gionalization of the primaxial domain, but rather with loss of the abaxialskeleton and increases in vertebral numbers independent of primaxialHox domain boundaries. We recommend that future studies of theorigin of the snake-like body form concentrate on elucidating the devel-opmental mechanisms by which the primaxial and abaxial skeletonsbecome dissociated18,24. The lateral somitic frontier is recognized as theboundary between the two developmental systems25, and we hypothe-size that future mapping of that frontier will demonstrate that majorinnovations in squamate body form are the result of abaxial modifica-tion whereas primaxial regionalization is conserved across amniotes,and potentially across vertebrates.
Online Content Methods, along with any additional Extended Data display itemsandSourceData, are available in the online version of the paper; references uniqueto these sections appear only in the online paper.
Received 30 July; accepted 4 November 2014.
Published online 5 January 2015.
1. Favier, B. & Dolle, P.Developmental functions of mammalianHox genes. Mol. Hum.Reprod. 3, 115–131 (1997).
2. Burke, A. C., Nelson, C. E., Morgan, B. A. & Tabin, C. Hox genes and the evolution ofvertebrate axial morphology. Development 121, 333–346 (1995).
3. Wellik, D. M. & Capecchi, M. R. Hox10 and Hox11 genes are required to globallypattern the mammalian skeleton. Science 301, 363–367 (2003).
4. Wellik, D. M. Hox patterning of the vertebrate skeleton. Dev. Dyn. 236, 2454–2463(2007).
5. McIntyre, D. C. et al. Hox patterning of the vertebrate rib cage. Development 134,2981–2989 (2007).
6. Carapuço, M., Novoa, A., Bobola, N. & Mallo, M. Hox genes specify vertebral types inthe presomitic mesoderm. Genes Dev. 19, 2116–2121 (2005).
7. Vinagre, T. et al. Evidence for a myotomal Hox/Myf cascade governingnonautonomous control of rib specification with global vertebral domains. Dev.Cell 18, 655–661 (2010).
8. Gaunt, S. J. Conservation in the Hox code during morphological evolution. Int.J. Dev. Biol. 38, 549–552 (1994).
9. Cohn, M. J.& Tickle, C.Developmental basis of limblessness and axial patterning insnakes. Nature 399, 474–479 (1999).
10. Woltering, J. M. et al. Axial patterning in snakes and caecilians: evidence for analternative interpretation of the Hox code. Dev. Biol. 332, 82–89 (2009).
11. Woltering, J. M. From lizard to snake; behind the evolution of an extreme bodyplan. Curr. Genomics 13, 289–299 (2012).
12. Di-Poı, N. et al. Changes in Hox genes’ structure and function during the evolutionof the squamate body plan. Nature 464, 99–103 (2010).
13. Guerreiro, I. et al. Role of a polymorphism in a Hox/Pax-responsive enhancer in theevolution of the vertebrate spine. Proc. Natl Acad. Sci. USA 110, 10682–10686(2013).
14. Muller, J. et al. Homeotic effects, somitogenesis and the evolution of vertebralnumbers in recent and fossil amniotes. Proc. Natl Acad. Sci. USA 107, 2118–2123(2010).
15. Gomez, C. et al. Control of segment number in vertebrate embryos. Nature 454,335–339 (2008).
16. Hoffstetter, R. & Gasc, J. P. in Biology of the Reptilia (eds Gans, C., Bellair, A. d’A. &Parsons, T. S.) Vol. 1, 201–310 (Academic, 1969).
17. Burke, A. C. & Nowicki, J. L. A new view of patterning domains in the vertebratemesoderm. Dev. Cell 4, 159–165 (2003).
18. Buchholtz, E. A. & Stepien, C. C. Anatomical transformation in mammals:developmental origin of aberrant cervical anatomy in tree sloths. Evol. Dev. 11,69–79 (2009).
19. Polly, P. D. & Head, J. J. in Morphometrics—Applications in Biology and Paleontology(ed. Elewa, A. M. T.) 197–222 (Springer, 2004).
20. Mansfield, J. H. & Abzhanov, A. Hox expression in the American Alligator andevolution of archosaurian axial patterning. J. Exper. Zool. B Mol. Dev. Evol. 314,629–644 (2010).
21. Shine, R. Vertebral numbers in male and female snakes: the roles of natural,sexual, and fecundity selection. J. Evol. Biol. 13, 455–465 (2000).
22. Prince, V. E., Joly, L., Ekker, M. & Ho, R. K. Zebrafish hox genes: genomicorganization and modified colinear expression patterns in the trunk. Development125, 407–420 (1998).
23. Mallo, M., Wellik, D. M. & Deschamps, J. Hox genes and regional patterning of thevertebrate body plan. Dev. Biol. 344, 7–15 (2010).
24. Shearman, R. M. & Burke, A. C. The lateral somatic frontier in ontogeny andphylogeny. J. Exp. Zool. B Mol. Dev. Evol. 312, 603–612 (2009).
25. Nowicki, J. L., Takimoto, R. & Burke, A. C. The lateral somitic frontier: dorso-ventralaspects of anterio-posterior reigonalization in avian embryos. Mech. Dev. 120,227–240 (2003).
Supplementary Information is available in the online version of the paper.
Acknowledgements We thank K. DeQueiroz, G. Zug, R. McDiarmid, K. Seymour,D. Gower, C. McCarthy, C. Bell, H. Voris, C. J. Cole, P.Holroyd andT. Labedz for specimenaccess, A. K. Behrensmeyer for access to microscopy facilities, and A. Goswami,K. Johnson, P. Mitteroecker, R. Raff, R. Reisz and M. Rowe for useful comments anddiscussion. This work was supported in part by a US National Science FoundationPostdoctoral Fellowship in Biological Informatics (DBI-0204082) to J.J.H., a NaturalSciences and Engineering Research Council of Canada Discovery Grant to J.J.H., and aUS National Science Foundation Grant (EAR-0843935) to P.D.P.
Author Contributions J.J.H. and P.D.P. designed the study. J.J.H. and P.D.P. collectedmorphometric data. J.J.H. and P.D.P. conducted morphometric analysis. P.D.P.designed and conducted segmented linear regression and maximum-likelihoodanalyses. J.J.H. and P.D.P. prepared figures and wrote the manuscript.
Author Information Morphometric data have been deposited in Dryad (http://dx.doi.org/10.5061/dryad.jq285). Reprints and permissions information is availableat www.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper. Correspondenceand requests for materials should be addressed to J.J.H. ([email protected]) orP.D.P. ([email protected]).
LETTER RESEARCH
G2015 Macmillan Publishers Limited. All rights reserved
2 A P R I L 2 0 1 5 | V O L 5 2 0 | N A T U R E | 8 9
METHODSMorphometric analysis. We quantified morphological regionalization using prin-cipal component shape variables derived from Procrustes superimposition26 oftwo-dimensional landmarks on vertebrae in anterior view19. We chose a taxonomicsample that covers all major squamate body forms and major clades (n 5 54 spec-imens; Extended Data Table 1). We selected anterior view because the centrum,neural arch and apophyses could all be identified by homologous landmarks. Thelandmarks we selected (Fig. 1 and Extended Data Table 2) define the aforementionedstructures, and are homologous among sampled squamates. For anatomical regionswhere distinct dorsal ribs are fused with vertebral apophyses (the ultimate pre-sacralvertebra in Pogona, Physignathus and Cordylus; Fig. 1), landmarks for the diapo-physis and parapophysis were placed at the dorsal and ventral edges of points offusion. In Alligator and Mus, we modified the landmarks for their taxon-specificvertebral morphologies (Extended Data Fig. 3 and Extended Data Table 2; see Sup-plementary Information for discussion and references). Because vertebrae are approx-imately bilaterally symmetrical, we digitized only the left side and midline of eachspecimen19. We omitted the atlas–axis complex from all analyses because it is adistinct anatomical system common to all amniotes. We used two-dimensional land-marks to make our data set more applicable for future analyses of fossil specimenswhose breakage and flattening frequently prohibits accurate three-dimensionalanalysis. Scores from the first five components, which represent more than 90% ofshape differences along the anterior–posterior axis for all taxa, were used as shapevariables for maximum-likelihood analyses of regionalization. To minimize theeffects of ontogenetic variation, we only sampled somatically mature specimensfor each taxon.Sampling along the anterior–posterior axis. For most analyses we used a stan-dardized sampling strategy in which we collected shape data at 5% intervals alongthe anterior–posterior axis beginning with the first post-atlanto-axial vertebra. If avertebra at a sampling point was pathologically or teratologically malformed, wesampled the next normally shaped element. This strategy facilitates comparisonsof regional models between taxa with radically different numbers of vertebrae, butdoes not allow the correspondence between morphological regions and Hox expres-sion boundaries to be assessed at the level of individual segments. To make directcomparisons between morphometric regions and Hox expression boundaries, wesampled the complete pre-cloacal column for key taxa.Intracolumnar shape variance. We measured intracolumnar shape variation foreach individual specimen as the total variance of Procrustes coordinates amongthe pre-cloacal vertebrae. Total variance is expected to be higher in taxa with moreregional differentiation. We used a permutation test to determine whether intra-columnar variance in squamate vertebral shape was significantly different in limbedversus snake-like taxa and in taxa identified as having four versus three regions. Toadjust for biases related to imbalance in the number of taxa in each category andnon-normal distribution of variances, we used a non-parametric permutation testin which the observed difference in the average variance in each group was comparedto a distribution of the same statistic calculated from 10,000 random permutations oftaxa between groups. Limbed taxa had significantly greater intervertebral variance(limbed 5 0.0133, snake-like 5 0.0095, P 5 0.0037), but taxa with four regions werenot significantly more variable than taxa with three regions (four region 5 0.0103,three region 5 0.0100, P 5 0.8245).Likelihood models of regionalization. Our analysis treats vertebral column regionsas a series of morphological gradients. Vertebrae within a region are not expectedto be identical: adjoining vertebrae spanning the boundary of two regions may bemore similar than each is to its opposite regional end member. Standard clusteranalysis is therefore inappropriate because it recovers hierarchical patterns ofvariation, not gradients of similarity. Our approach uses segmented linear regres-sion (SLR)27,28 on the first five PC scores to recover gradients of morphology andthe breaks between them (Extended Data Fig. 2). In SLR, a series of contiguousregression lines are fit to the data such that each segment has its own slopes andintercepts. Boundaries are estimated by finding the break points that minimize theresidual sum of squares, adding an additional parameter to the model for each pairof segments. In our models, each segment of the regression therefore correspondsto a morphological region, its slope(s) describe its shape gradient, and the breakpoints correspond to the regional boundaries.
To estimate the number of regions and the positions of regional boundaries, weiteratively fit four classes of model with one, two, three and four segments, respec-tively, to each vertebral column. The classes correspond to a morphological spec-trum from complete deregionalization (one segment) to hyper-regionalized (foursegments: cervical, anterior thoracic, posterior thoracic, lumbar). Each class of modelhas many specific instances that differ in the slope of the regression segments andthe position of the boundaries between them (Extended Data Fig. 2). The likeli-hood of each instance of each model was assessed using a likelihood ratio, which inits general form is:
l(x)~L(h0 x)jL(h1 x)j ð1Þ
where l(x) is the likelihood of hypothesis H0 relative to hypothesis H1 (the hypo-theses in our case are different models of regionalization), L(h0 x)j is the likelihoodof h0, which are the parameters of the H0 model given the data (which in our caseare the vertebral shape scores), and L(h1 x)j is the likelihood of the H1 model param-eters. Specifically, we calculated the log- likelihood ratio test statistic, D, for oursegmented regressions using the residual sum of squares (RSS) as follows:
D~{2ln(l)~nln(S0=S1) ð2Þ
where l is the likelihood ratio, n is the number of data points (five times the numberof vertebrae since we used scores from the first five dimensions of our vertebralshape spaces), and S0 and S1 are the RSS for H0 and H1, respectively29. The regres-sion slopes and intercepts were found by exact calculation (the parameters that max-imize the likelihood function are the same as those found by least-squares fitting).We used a grid search to calculate the likelihood of every possible set of regionalbreaks, thus providing us with a complete statistical distribution for testing alter-native regional models29.Model selection using AICc. The likelihoods of models with different numbers ofregions are not directly comparable because the number of parameters differ. Ourmodels have 10k 1 k 2 1 parameters for each of their k regions: one slope and inter-cept for each of the five dimensions in each region plus one boundary between eachregion (Extended Data Fig. 2). Our one-region model class has 10 parameters, thetwo-region class has 21 parameters, the three-region class has 62, and the four-regionclass has 83. The more parameters a model has, the better it fits the data (when thenumber of regions increases to equal the number of vertebrae it will always fit thedata perfectly). Model comparisons thus require an adjustment for the number ofparameters, especially when comparing taxa with different numbers of vertebrae(a 20-region model will fit a 20-vertebrae lizard column perfectly, but it will not fita 200-vertebrae snake column as well). We used the corrected AICc to adjust thelikelihood ratios by the number of model parameters and data points so that theycould be compared between model classes to objectively select the best model ofregionalization:
AICc~D{2(pz1)n
n{p{2ð3Þ
where D is the log-likelihood ratio from equation (2), p is the number of param-eters (10k 1 k 2 1 for our study, where k is the number of regions), and n is thenumber of data points (five times the number of vertebrae in our study). This cor-rection penalizes the log likelihood for each additional parameter and makes thepenalty proportionally heavier for smaller data sets. This value is scaled so that thebest model is the one with the highest AICc value.Testing hypotheses of evolutionary changes in regionalization. We assessed therelative support for four competing hypotheses of evolutionary changes in region-alization in squamates and the ancestral state of regionalization in snake-like forms.For each hypothesis, the expected number of regions was mapped onto phylogeny(Fig. 2d). Total support for each hypothesis was estimated as the sum of the AICc
support values for the best corresponding regional model for each taxon (ExtendedData Table 3). For example, support for the hypothesis that all reptiles have twovertebral regions can be calculated by summing the AICc values for the best two-region model of all of species in the analysis. The highest possible support for anysuch hypothesis occurs when each taxon is assigned the number of regions that isbest supported by its own data (Fig. 2d, left bar). The sum of the AICc values is notmeaningful when taxa vary in the number of vertebrae, so we used the data sampledat 5% intervals, which have a maximum summed AICc support of 28,140.6. Notethat, although this measure of total support varies with the number of taxa includedin an analysis, the number of taxa is constant across the four hypotheses; relativesupport for the competing evolutionary hypotheses can therefore be measured asthe fractional difference of the summed AICc values of each model relative to thetotal AICc of the best- and worst-supported hypothesis (1.0 5 best, 0.0 5 worst)30
.
Comparison of morphometric boundaries to boundaries of Hox expression.Because one of our aims is to determine whether morphological regionalization cor-responds to Hox gene expression domains, it is necessary to test statistically whetherthe morphometric regional boundaries differ from expression boundaries. Ourlikelihood framework allows the relative support of the best morphometric regionalmodel to be compared to other models. The probability that an alternative set ofboundaries differs from the best morphometric model was assessed by countingvalues in which the fit was better than or equal to the alternative model and nor-malizing by the number of possible regional models. The resulting P value is the
RESEARCH LETTER
G2015 Macmillan Publishers Limited. All rights reserved
probability that expression boundaries differ from the best regional model (ExtendedData Fig. 5).
All calculations were performed using Mathematica v.9.0.
26. Rohlf, F. J. & Slice, D. Extensions of the Procrustes method for the optimalsuperimposition of landmarks. Syst. Biol. 39, 40–59 (1990).
27. Hudson, D. Fitting segmented curves whose join points have to be estimated.J. Am. Stat. Assoc. 61, 1097–1129 (1966).
28. Feder,P.The log likelihoodratio insegmentedregression.Ann.Stat.3,84–97(1975).29. Lerman, P. Fitting segmented regression models by grid search. Appl. Stat. 29,
77–84 (1980).30. Claeskens, G. & Hjort, N. Model Selection and Model Averaging (Cambridge Univ,
Press, 2008).
LETTER RESEARCH
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Figure 1 | Skeletal morphology and intracolumnar shapevariation in the pre-cloacal vertebral column of limbed lizards andsnakes. a, Skeleton of limbed lizard (Pogona minor) in dorsal view. b, Skeletonof snake (Hypsiglena torquata) in dorsal view. c, Principal component analysis(PCA) ordination of pre-cloacal vertebral shape variables derived fromgeometric morphometric analysis in a limbed lizard (Pogona vitticeps) based on
first two principal components (PC 1 and PC 2). d, PCA ordination ofpre-cloacal vertebral shape variables in a snakes (Pantherophis guttatus).Ordination using the first two components describes intracolumnar shapechange along the anterior–posterior axis of the pre-cloacal vertebral columnand explains .90% of overall shape variation.
RESEARCH LETTER
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Figure 2 | Model fitting with segmented linear regression.a–f, A series of regional models were fit to each taxon using a series ofsegmented linear regressions. In each case vertebral shape variables (orangedots) were regressed onto position in the vertebral column (brown lines).Models differ in both the number of regions and the position of regionalboundaries. a–f, Two examples are shown for each of two, three and fourregions, where the right column shows the best fitting example for each. Redarrows mark the regional boundaries in each example. The slope of eachsegment (heavy dark line) represents the shape gradient each region and the
residual sum of squares (RSS) represents the lack of fit of the model to the data.f, The model with the highest likelihood. The log likelihood of each model isproportional to this model. However, the number of parameters increaseswith the number of regions, as does the likelihood of the model; thereforecorrected Akaike adjustment (AICc) is required to select the best model. b, Thebest model using AICc. It is the two-region model with the breakpoint 25%along the pre-cloacal vertebral column. This example is based on the firstprincipal component of Eunectes notaeus.
LETTER RESEARCH
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Figure 3 | Morphometric landmarks used to quantifyprimaxial shape variance and regionalization in pre-cloacal vertebrae ofMus and Alligator. a, b, Elements for both Mus (a) and Alligator (b) are,from top to bottom: first post-atlanto-axial vertebrae, third thoracic (Mus) and
sixth dorsal vertebrae (Alligator), last lumbar vertebrae. See Extended DataTable 2 and Supplementary Information for description of landmarks anddiscussion of landmark selection.
RESEARCH LETTER
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Figure 4 | Best-fit regionalization models for completepre-cloacal skeletons of Mus, Alligator and select squamates. AICc scores arereported for the best regional model from each taxon. Taxa in bold are snakes or
have snake-like body forms. Cells represent individual vertebrae in each regionfor the complete pre-cloacal/pre-sacral vertebral column in each taxon. Cellcolours represent morphometric regions. C, cervical; L, lumbar; T, thoracic.
LETTER RESEARCH
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Figure 5 | Comparison of best-fit four-region model withmodels fitting morphometric regional boundaries to expression boundariesfor Hox10 genes. a, Best-fit model. b, Fit to anterior expression boundaries ofHoxA10 and HoxC10 from ref. 12. c, Fit to anterior expression boundary forHoxC10 from ref. 10. d, Fit to posterior expression boundaries of HoxA10 andHoxC10 from ref. 12. Abbreviations are the same as for Fig. 3. Only theposterior boundaries of Hox10 expression are significantly worse fits than thebest-fit model. RSS, residual sum of squares for segmented linear regression.P values are probability that the Hox boundaries differ from the bestregional model.
RESEARCH LETTER
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Table 1 | Examined specimens
AMNH, American Museum of Natural History, New York; BMNH-R, The Natural History Museum, London; FMNH, Field Museum of Natural History; IU, Indiana University; ROMV-R, Royal Ontario Museum RecentVertebrate Collection; TMM, Texas Memorial Museum, University of Texas at Austin; UCMP, University of California Museum of Paleontology; UNL ZM, University of Nebraska—Lincoln, Museum of Zoology; USNM,United States National Museum, Smithsonian Institution.*Specimens not used in morphometric analysis.
LETTER RESEARCH
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Table 2 | Landmarks and corresponding morphology used in morphometric analysis
Landmarks document intracolumnar variation in vertebral shape for squamates, Alligator and Mus. For discussion and references, see Supplementary Information.
RESEARCH LETTER
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Table 3 | AICc values for regionalization models
Dose ID Day post infusion
3BNC117 IC50 ( g/ml) 3BNC117 IC50
Clone Cloning procedure
Vector backbone
3BNC117 (IC50; g/ml)
Average (geo. mean)
Day 0 NDDay 28 0.90
Day 0 0.11Day 28 3.78
Day 0 0.07Day 28 0.94
Day 0 0.78Day 28 >20
Day 0 >20Day 28 ND
Day 0 0.20Day 28 0.30
Day 0 0.49Day 56 0.03
Day 0 >20Day 28 >20
2C5_D0_12 gp120 pSVIII 0.0152C5_D0_21 gp120 pSVIII 0.0172C5_D0_27 gp120 pSVIII 0.0172C5_W4_59 gp120 pSVIII 11.5432C5_W4_22 gp120 pSVIII 6.7372C5_W4_27 gp120 pSVIII 7.5142C5_W4_28 gp120 pSVIII 3.4952C5_W4_34 gp120 pSVIII 8.758
2C1_D0_12 gp120 pSVIII 0.2092C1_D0_22 gp120 pSVIII 0.1582C1_D0_32 gp120 pSVIII 0.0062C1_W4_12 gp120 pSVIII 0.0432C1_W4_18 gp120 pSVIII 0.2252C1_W4_31 gp120 pSVIII 0.262
2D1_D0_D5 gp160 pcDNA3.1 0.1652D1_D0_B3.1 gp160 pcDNA3.1 0.1282D1_D0_B10 gp160 pcDNA3.1 0.1722D1_W4_37 gp120 pSVIII 0.5782D1_W4_40 gp120 pSVIII 0.5012D1_W4_69 gp120 pSVIII 0.4652D1_W4_71 gp120 pSVIII 0.523
Day 0 0.13Day 28 0.35
2E1_D0_12 gp160 pcDNA3.1 0.1032E1_D0_20 gp160 pcDNA3.1 0.1152E1_D0_34 gp160 pcDNA3.1 0.0682E1_W4_23 gp160 pcDNA3.1 0.0412E1_W4_E1 gp160 pcDNA3.1 0.5902E1_W4_F6 gp160 pcDNA3.1 0.496
2E2_D0_A10 gp160 pcDNA3.1 0.0172E2_D0_C3 gp160 pcDNA3.1 0.0102E2_D0_E9 gp160 pcDNA3.1 0.0182E2_W4_B9 gp160 pcDNA3.1 0.017
2E2_W4_C11 gp160 pcDNA3.1 0.0202E2_W4_D5 gp160 pcDNA3.1 0.057
Day 0 0.18Day 28 1.10
Day 0 0.24Day 28 ND
Day 0 0.30Day 28 ND
Autologous virus isolates
ND
ND
15.36
167.0
7.09
0.54
1.78
0.18
2.24
0.06
0.02
1.8
0.14
0.09
0.90
Day 0
Day 28
Day 0
2D1
Day 28
Day -7
2C5
2C1
ND
Day 28
2E1
0.68
Day 28
ND
1.5
0.1
No change
ND
ND
ND
ND
ND
0.15
12.3
0.01
0.03
ND
0.09
0.23
1 m
g/kg
3 m
g/kg
2B1
2B2
2B3
2A1
10 m
g/kg
2E4 ND
2E5 ND
30 m
g/kg
2E3
2.7
6.1
ND
0.40Day 0
Day 28
Day 0
2E2
HIV-1 envelopes cloned from plasma
2C2
2C4
2D3
2A3
2A4
ND
35.3
13.5
25.9
ND
ND
ND
ND
1.3
0.52
Values are for models from one to four morphological regions through the pre-sacral/pre-cloacal vertebral columns of select amniotes sampled at 5% intervals along the anterior–posterior axis.
LETTER RESEARCH
G2015 Macmillan Publishers Limited. All rights reserved
Extended Data Table 4 | AICc values for regionalization models
Values are for models from one to four morphological regions through the complete pre-sacral/pre-cloacal vertebral columns of select amniotes.
RESEARCH LETTER
G2015 Macmillan Publishers Limited. All rights reserved
