Journal of Molecular Evolution

, Volume 67, Issue 6, pp 581–593 | Cite as

A Molecular Footprint of Limb Loss: Sequence Variation of the Autopodial Identity Gene Hoxa-13

  • Tiana Kohlsdorf
  • Michael P. Cummings
  • Vincent J. Lynch
  • Geffrey F. Stopper
  • Kazuhiko Takahashi
  • Günter P. Wagner


The homeobox gene Hoxa-13 codes for a transcription factor involved in multiple functions, including body axis and hand/foot development in tetrapods. In this study we investigate whether the loss of one function (e.g., limb loss in snakes) left a molecular footprint in exon 1 of Hoxa-13 that could be associated with the release of functional constraints caused by limb loss. Fragments of the Hoxa-13 exon 1 were sequenced from 13 species and analyzed, with additional published sequences of the same region, using relative rates and likelihood-ratio tests. Five amino acid sites in exon 1 of Hoxa-13 were detected as evolving under positive selection in the stem lineage of snakes. To further investigate whether there is an association between limb loss and sequence variation in Hoxa-13, we used the random forest method on an alignment that included shark, basal fish lineages, and “eu-tetrapods” such as mammals, turtle, alligator, and birds. The random forest method approaches the problem as one of classification, where we seek to predict the presence or absence of autopodium based on amino acid variation in Hoxa-13 sequences. Different alignments tested were associated with similar error rates (18.42%). The random forest method suggested that phenotypic states (autopodium present and absent) can often be correctly predicted based on Hoxa-13 sequences. Basal, nontetrapod gnathostomes that never had an autopodium were consistently classified as limbless together with the snakes, while eu-tetrapods without any history of limb loss in their phylogeny were also consistently classified as having a limb. Misclassifications affected mostly lizards, which, as a group, have a history of limb loss and limb re-evolution, and the urodele and caecilian in our sample. We conclude that a molecular footprint can be detected in Hoxa-13 that is associated with the lack of an autopodium; groups with classification ambiguity (lizards) are characterized by a history of repeated limb loss and possible limb re-evolution.


Hoxa-13 Molecular evolution Limb loss Squamata 



The authors thank K. Crow for help with lab molecular techniques and discussion of results, and Jacques Gauthier and Greg Watkins-Colwell (Yale Peabody Museum), Adalgisa Caccono (Yale University EEB), and David Wake (Museum of Vertebrate Zoology, University of California, Berkeley) for donation of tissue samples. We thank Drs. H. Greene, J. Gauthier, and P. Weldon for discussion of morphological patterns in snakes, Dr. K. Schwenk for discussion of phylogenetic relationships in Squamata, and M. Conte for assistance with some of the analyses. T.K. was supported by a CNPq/Brazil postdoctoral fellowship (PDE-201244/2003-9) and is supported by a FAPESP/Brazil grant (05/60140-4); G.P.W. is supported by a NSF/USA grant (IOB-0445971).

Supplementary material

239_2008_9156_MOESM1_ESM.doc (40 kb)
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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Tiana Kohlsdorf
    • 1
    • 2
  • Michael P. Cummings
    • 3
  • Vincent J. Lynch
    • 1
  • Geffrey F. Stopper
    • 1
  • Kazuhiko Takahashi
    • 1
    • 4
  • Günter P. Wagner
    • 1
  1. 1.Department of Ecology and Evolutionary BiologyYale UniversityNew HavenUSA
  2. 2.Departamento de BiologiaFFCLRP, University of São PauloRibeirao PretoBrazil
  3. 3.Center for Bioinformatics and Computational BiologyUniversity of MarylandCollege ParkUSA
  4. 4.National Institute for Basic BiologyOkazaki National Research InstitutesOkazakiJapan

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