Evolution of the snake body form reveals homoplasy in amniote Hox gene function

Journal name:
Nature
Volume:
520,
Pages:
86–89
Date published:
DOI:
doi:10.1038/nature14042
Received
Accepted
Published online

Hox genes regulate regionalization of the axial skeleton in vertebrates1, 2, 3, 4, 5, 6, 7, and changes in their expression have been proposed to be a fundamental mechanism driving the evolution of new body forms8, 9, 10, 11, 12, 13, 14. The origin of the snake-like body form, with its deregionalized pre-cloacal axial skeleton, has been explained as either homogenization of Hox gene expression domains9, or retention of standard vertebrate Hox domains with alteration of downstream expression that suppresses development of distinct regions10, 11, 12, 13. Both models assume a highly regionalized ancestor, but the extent of deregionalization of the primaxial domain (vertebrae, dorsal ribs) of the skeleton in snake-like body forms has never been analysed. Here we combine geometric morphometrics and maximum-likelihood analysis to show that the pre-cloacal primaxial domain of elongate, limb-reduced lizards and snakes is not deregionalized compared with limbed taxa, and that the phylogenetic structure of primaxial morphology in reptiles does not support a loss of regionalization in the evolution of snakes. We demonstrate that morphometric regional boundaries correspond to mapped gene expression domains in snakes, suggesting that their primaxial domain is patterned by a normally functional Hox code. Comparison of primaxial osteology in fossil and modern amniotes with Hox gene distributions within Amniota indicates that a functional, sequentially expressed Hox code patterned a subtle morphological gradient along the anterior–posterior axis in stem members of amniote clades and extant lizards, including snakes. The highly regionalized skeletons of extant archosaurs and mammals result from independent evolution in the Hox code and do not represent ancestral conditions for clades with snake-like body forms. The developmental origin of snakes is best explained by decoupling of the primaxial and abaxial domains and by increases in somite number15, not by changes in the function of primaxial Hox genes9, 10.

At a glance

Figures

  1. Morphological variation in the pre-cloacal vertebral column of limbed lizards and snakes.
    Figure 1: Morphological variation in the pre-cloacal vertebral column of limbed 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 landmarks shown on mid-trunk vertebra of Pogona were used to characterize vertebral shape (Extended Data Table 2).

  2. Regional boundaries, evolutionary models of regional changes, and intracolumnar variance.
    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, Box plot of intracolumnar variances in limbed (n = 10 specimens) and snake-like (n = 42 specimens) squamates. c, Regional boundaries in primaxial domains for each taxon subsampled at 5% intervals. Coloured cells represent vertebrae in different regions of the best-fit model, for which corrected Akaike information criterion (AICc) scores are given. Taxa in bold are snakes and snake-like squamates. d, Best-fit distribution of regions (left) compared with four models for evolutionary changes in regionalization. Each is depicted by the number of regions (2 to 4) expected in limbed and snake-like taxa. RS, relative support (Methods).

  3. Correspondence between Hox expression domains and morphometric boundaries for four-region models of primaxial regionalization.
    Figure 3: Correspondence between Hox expression domains and morphometric boundaries for four-region models of primaxial regionalization.

    Coloured bars represent expression domains for Mus4, 20 and Alligator20, and the range of anterior expression boundaries for Pantherophis10, 11, 12. Hox expression domains for the thoracic–lumbar transition in Alligator have not yet been mapped20. Cells represent individual vertebrae in each region for the entire pre-cloacal/pre-sacral vertebral column in each taxon. Cell colours represent morphometric regions. Grey bars indicate regions of overlap between genes and morphometric regions. C, cervical; L, lumbar; T, thoracic; V, vertebra.

  4. Time-calibrated phylogeny of selected extant and fossil amniotes, illustrating pre-cloacal and pre-sacral primaxial skeletal regionalization and the generalized ancestral amniote pattern of Hox expression.
    Figure 4: Time-calibrated phylogeny of selected extant and fossil amniotes, illustrating pre-cloacal and pre-sacral primaxial skeletal regionalization and the generalized ancestral amniote pattern of Hox expression.

    Node numbers 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. Coloured bars represent relative positions of anterior expression domain boundaries for Hox4–10 paralogues along the anterior–posterior axis in Amniota. See Supplementary Information for data sources. Cen., Cenozoic; Pal., Palaeozoic. Daggers indicate fossil taxa.

  5. Skeletal morphology and intracolumnar shape variation in the pre-cloacal vertebral column of limbed lizards and snakes.
    Extended Data Fig. 1: Skeletal morphology and intracolumnar shape variation in the pre-cloacal vertebral column of limbed lizards and snakes.

    a, Skeleton of limbed lizard (Pogona minor) in dorsal view. b, Skeleton of snake (Hypsiglena torquata) in dorsal view. c, Principal component analysis (PCA) ordination of pre-cloacal vertebral shape variables derived from geometric morphometric analysis in a limbed lizard (Pogona vitticeps) based on first two principal components (PC 1 and PC 2). d, PCA ordination of pre-cloacal vertebral shape variables in a snakes (Pantherophis guttatus). Ordination using the first two components describes intracolumnar shape change along the anterior–posterior axis of the pre-cloacal vertebral column and explains >90% of overall shape variation.

  6. Model fitting with segmented linear regression.
    Extended Data Fig. 2: Model fitting with segmented linear regression.

    af, A series of regional models were fit to each taxon using a series of segmented linear regressions. In each case vertebral shape variables (orange dots) were regressed onto position in the vertebral column (brown lines). Models differ in both the number of regions and the position of regional boundaries. af, Two examples are shown for each of two, three and four regions, where the right column shows the best fitting example for each. Red arrows mark the regional boundaries in each example. The slope of each segment (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 is proportional to this model. However, the number of parameters increases with the number of regions, as does the likelihood of the model; therefore corrected Akaike adjustment (AICc) is required to select the best model. b, The best 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 first principal component of Eunectes notaeus.

  7. Morphometric landmarks used to quantify primaxial shape variance and regionalization in pre-cloacal vertebrae of Mus and Alligator.
    Extended Data Fig. 3: Morphometric landmarks used to quantify primaxial shape variance and regionalization in pre-cloacal vertebrae of Mus 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 Data Table 2 and Supplementary Information for description of landmarks and discussion of landmark selection.

  8. Best-fit regionalization models for complete pre-cloacal skeletons of Mus, Alligator and select squamates.
    Extended Data Fig. 4: Best-fit regionalization models for complete pre-cloacal skeletons of Mus, Alligator and select squamates.

    AICc scores are reported 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 region for the complete pre-cloacal/pre-sacral vertebral column in each taxon. Cell colours represent morphometric regions. C, cervical; L, lumbar; T, thoracic.

  9. Comparison of best-fit four-region model with models fitting morphometric regional boundaries to expression boundaries for Hox10 genes.
    Extended Data Fig. 5: Comparison of best-fit four-region model with models fitting morphometric regional boundaries to expression boundaries for Hox10 genes.

    a, Best-fit model. b, Fit to anterior expression boundaries of HoxA10 and HoxC10 from ref. 12. c, Fit to anterior expression boundary for HoxC10 from ref. 10. d, Fit to posterior expression boundaries of HoxA10 and HoxC10 from ref. 12. Abbreviations are the same as for Fig. 3. Only the posterior boundaries of Hox10 expression are significantly worse fits than the best-fit model. RSS, residual sum of squares for segmented linear regression. P values are probability that the Hox boundaries differ from the best regional model.

Tables

  1. Examined specimens
    Extended Data Table 1: Examined specimens
  2. Landmarks and corresponding morphology used in morphometric analysis
    Extended Data Table 2: Landmarks and corresponding morphology used in morphometric analysis
  3. AICc values for regionalization models
    Extended Data Table 3: AICc values for regionalization models
  4. AICc values for regionalization models
    Extended Data Table 4: AICc values for regionalization models

References

  1. Favier, B. & Dollé, P. Developmental functions of mammalian Hox genes. Mol. Hum. Reprod. 3, 115131 (1997)
  2. Burke, A. C., Nelson, C. E., Morgan, B. A. & Tabin, C. Hox genes and the evolution of vertebrate axial morphology. Development 121, 333346 (1995)
  3. Wellik, D. M. & Capecchi, M. R. Hox10 and Hox11 genes are required to globally pattern the mammalian skeleton. Science 301, 363367 (2003)
  4. Wellik, D. M. Hox patterning of the vertebrate skeleton. Dev. Dyn. 236, 24542463 (2007)
  5. McIntyre, D. C. et al. Hox patterning of the vertebrate rib cage. Development 134, 29812989 (2007)
  6. Carapuço, M., Novoa, A., Bobola, N. & Mallo, M. Hox genes specify vertebral types in the presomitic mesoderm. Genes Dev. 19, 21162121 (2005)
  7. Vinagre, T. et al. Evidence for a myotomal Hox/Myf cascade governing nonautonomous control of rib specification with global vertebral domains. Dev. Cell 18, 655661 (2010)
  8. Gaunt, S. J. Conservation in the Hox code during morphological evolution. Int. J. Dev. Biol. 38, 549552 (1994)
  9. Cohn, M. J. & Tickle, C. Developmental basis of limblessness and axial patterning in snakes. Nature 399, 474479 (1999)
  10. Woltering, J. M. et al. Axial patterning in snakes and caecilians: evidence for an alternative interpretation of the Hox code. Dev. Biol. 332, 8289 (2009)
  11. Woltering, J. M. From lizard to snake; behind the evolution of an extreme body plan. Curr. Genomics 13, 289299 (2012)
  12. Di-Poï, N. et al. Changes in Hox genes’ structure and function during the evolution of the squamate body plan. Nature 464, 99103 (2010)
  13. Guerreiro, I. et al. Role of a polymorphism in a Hox/Pax-responsive enhancer in the evolution of the vertebrate spine. Proc. Natl Acad. Sci. USA 110, 1068210686 (2013)
  14. Müller, J. et al. Homeotic effects, somitogenesis and the evolution of vertebral numbers in recent and fossil amniotes. Proc. Natl Acad. Sci. USA 107, 21182123 (2010)
  15. Gomez, C. et al. Control of segment number in vertebrate embryos. Nature 454, 335339 (2008)
  16. Hoffstetter, R. & Gasc, J. P. in Biology of the Reptilia (eds Gans, C., Bellair, A. d’A. & Parsons, T. S.) Vol. 1 201310 (Academic, 1969)
  17. Burke, A. C. & Nowicki, J. L. A new view of patterning domains in the vertebrate mesoderm. Dev. Cell 4, 159165 (2003)
  18. Buchholtz, E. A. & Stepien, C. C. Anatomical transformation in mammals: developmental origin of aberrant cervical anatomy in tree sloths. Evol. Dev. 11, 6979 (2009)
  19. Polly, P. D. & Head, J. J. in Morphometrics—Applications in Biology and Paleontology (ed. Elewa, A. M. T.) 197222 (Springer, 2004)
  20. Mansfield, J. H. & Abzhanov, A. Hox expression in the American Alligator and evolution of archosaurian axial patterning. J. Exper. Zool. B Mol. Dev. Evol. 314, 629644 (2010)
  21. Shine, R. Vertebral numbers in male and female snakes: the roles of natural, sexual, and fecundity selection. J. Evol. Biol. 13, 455465 (2000)
  22. Prince, V. E., Joly, L., Ekker, M. & Ho, R. K. Zebrafish hox genes: genomic organization and modified colinear expression patterns in the trunk. Development 125, 407420 (1998)
  23. Mallo, M., Wellik, D. M. & Deschamps, J. Hox genes and regional patterning of the vertebrate body plan. Dev. Biol. 344, 715 (2010)
  24. Shearman, R. M. & Burke, A. C. The lateral somatic frontier in ontogeny and phylogeny. J. Exp. Zool. B Mol. Dev. Evol. 312, 603612 (2009)
  25. Nowicki, J. L., Takimoto, R. & Burke, A. C. The lateral somitic frontier: dorso-ventral aspects of anterio-posterior reigonalization in avian embryos. Mech. Dev. 120, 227240 (2003)
  26. Rohlf, F. J. & Slice, D. Extensions of the Procrustes method for the optimal superimposition of landmarks. Syst. Biol. 39, 4059 (1990)
  27. Hudson, D. Fitting segmented curves whose join points have to be estimated. J. Am. Stat. Assoc. 61, 10971129 (1966)
  28. Feder, P. The log likelihood ratio in segmented regression. Ann. Stat. 3, 8497 (1975)
  29. Lerman, P. Fitting segmented regression models by grid search. Appl. Stat. 29, 7784 (1980)
  30. Claeskens, G. & Hjort, N. Model Selection and Model Averaging (Cambridge Univ, Press, 2008)

Download references

Author information

Affiliations

  1. Department of Earth and Atmospheric Sciences and Nebraska State Museum of Natural History, University of Nebraska–Lincoln, Lincoln, Nebraska 68588-0340, USA

    • Jason J. Head
  2. Departments of Geological Sciences, Biology and Anthropology, Indiana University, Bloomington, Indiana 47405-1405, USA

    • P. David Polly

Contributions

J.J.H. and P.D.P. designed the study. J.J.H. and P.D.P. collected morphometric data. J.J.H. and P.D.P. conducted morphometric analysis. P.D.P. designed and conducted segmented linear regression and maximum-likelihood analyses. J.J.H. and P.D.P. prepared figures and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Morphometric data have been deposited in Dryad (http://dx.doi.org/10.5061/dryad.jq285).

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Skeletal morphology and intracolumnar shape variation in the pre-cloacal vertebral column of limbed lizards and snakes. (256 KB)

    a, Skeleton of limbed lizard (Pogona minor) in dorsal view. b, Skeleton of snake (Hypsiglena torquata) in dorsal view. c, Principal component analysis (PCA) ordination of pre-cloacal vertebral shape variables derived from geometric morphometric analysis in a limbed lizard (Pogona vitticeps) based on first two principal components (PC 1 and PC 2). d, PCA ordination of pre-cloacal vertebral shape variables in a snakes (Pantherophis guttatus). Ordination using the first two components describes intracolumnar shape change along the anterior–posterior axis of the pre-cloacal vertebral column and explains >90% of overall shape variation.

  2. Extended Data Figure 2: Model fitting with segmented linear regression. (379 KB)

    af, A series of regional models were fit to each taxon using a series of segmented linear regressions. In each case vertebral shape variables (orange dots) were regressed onto position in the vertebral column (brown lines). Models differ in both the number of regions and the position of regional boundaries. af, Two examples are shown for each of two, three and four regions, where the right column shows the best fitting example for each. Red arrows mark the regional boundaries in each example. The slope of each segment (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 is proportional to this model. However, the number of parameters increases with the number of regions, as does the likelihood of the model; therefore corrected Akaike adjustment (AICc) is required to select the best model. b, The best 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 first principal component of Eunectes notaeus.

  3. Extended Data Figure 3: Morphometric landmarks used to quantify primaxial shape variance and regionalization in pre-cloacal vertebrae of Mus and Alligator. (350 KB)

    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 Data Table 2 and Supplementary Information for description of landmarks and discussion of landmark selection.

  4. Extended Data Figure 4: Best-fit regionalization models for complete pre-cloacal skeletons of Mus, Alligator and select squamates. (323 KB)

    AICc scores are reported 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 region for the complete pre-cloacal/pre-sacral vertebral column in each taxon. Cell colours represent morphometric regions. C, cervical; L, lumbar; T, thoracic.

  5. Extended Data Figure 5: Comparison of best-fit four-region model with models fitting morphometric regional boundaries to expression boundaries for Hox10 genes. (371 KB)

    a, Best-fit model. b, Fit to anterior expression boundaries of HoxA10 and HoxC10 from ref. 12. c, Fit to anterior expression boundary for HoxC10 from ref. 10. d, Fit to posterior expression boundaries of HoxA10 and HoxC10 from ref. 12. Abbreviations are the same as for Fig. 3. Only the posterior boundaries of Hox10 expression are significantly worse fits than the best-fit model. RSS, residual sum of squares for segmented linear regression. P values are probability that the Hox boundaries differ from the best regional model.

Extended Data Tables

  1. Extended Data Table 1: Examined specimens (461 KB)
  2. Extended Data Table 2: Landmarks and corresponding morphology used in morphometric analysis (365 KB)
  3. Extended Data Table 3: AICc values for regionalization models (695 KB)
  4. Extended Data Table 4: AICc values for regionalization models (242 KB)

Supplementary information

PDF files

  1. Supplementary Information (172 KB)

    This file contains Supplementary Discussions 1-2, the data acquisition details for the methods, the data sources for the figures and additional references.

Additional data