Ongoing research

The adaptation of the tetrapods to a life on land was a great step in vertebrate evolution. Several fish groups have attempted to colonise the terrestrial environment (e.g. mudskippers among ray-finned fish; lungfish among lobe-finned fish) but only tetrapods were able to initiate a large-scale terrestrial radiation. This event, which occurred a bit less than 400 million years ago, has been a focus of interest for more than a century. Based on novel, non-destructive synchrotron-based techniques, we have imaged the detailed bone microstructure of key fossils in three dimensions and aim to address the following questions:

When did appendage elements acquire long bone features?
How could limb bones adapt to terrestrial conditions?
What can mineralised tissues tell us about the life history of early tetrapods?

When did appendage elements acquire long bone features?

A typical mammal long bone consists of a shaft terminated by articular condyles. Long-bone features were progressively acquired during the evolution of tetrapods. We want to determine this sequence through 3D imaging and modelling of fin and limb bones (from fish members of the tetrapod stem group and crown tetrapods). We study the time of appearance and original function of long-bone components and associated soft tissues. We focus on dating the establishment of endochondral ossification, growth plates, bone marrow processes and secondary centres of ossification in tetrapod long bones.

Figure 2: 3D virtual reconstruction of the humerus of a 380 million-year old fish, *Eusthenopteron*, showing inner longitudinal bone-marrow processes (in purple).

Selected publications:

Estefa J. 2014. The 3D limb-bone histology of Seymouria sanjuanensis sheds light on the long-bone growth and posture of terrestrial stem amniotes. Master project.

Sanchez S., Tafforeau P. & P. E. Ahlberg. 2014. The humerus of Eusthenopteron: a puzzling organization presaging the establishment of tetrapod limb bone marrow. Proceedings of the Royal Society B, 281-1782: 20140299.

Sanchez S., Ricqlès A. de, Schoch R. & J.S. Steyer. 2010. Developmental plasticity of limb bone microstructural organization in Apateon: histological evidence of paedomorphic conditions in branchiosaurs. Evolution and Development, 12-3: 315-328.

How could limb bones adapt to terrestrial conditions?

We want to understand the adaptation of limb bones to terrestrial conditions. Because the microanatomy of early-tetrapod limb bones significantly differs from that of extant tetrapods, we focus on testing the resistance of early-tetrapod microanatomy to terrestrial loading stresses using 3D modelling. We also aim to reconstruct the muscular architecture of early tetrapods through the development of new tomographic techniques in collaboration with the European Synchrotron Radiation Facility (France).

Figure 3: Comparative series of virtual thin sections through the humerus of the extant salamander *Desmognathus* (pointed out with the red arrow) showing fibres embedded in the bone at the location of a muscle attachment.

Selected publications:

Estefa J. 2014. The 3D limb-bone histology of Seymouria sanjuanensis sheds light on the long-bone growth and posture of terrestrial stem amniotes. Master project.

Sanchez S., Dupret V., Tafforeau P., Trinajstic K. M., Ryll B., Gouttenoire P.-J., Wretman L., Zylberberg L., Peyrin F. & Per E. Ahlberg. 2013. 3D microstructural architecture of muscle attachments in extant and fossil vertebrates revealed by synchrotron microtomography. PloS One, 8-2: e56992.

Sanchez S., Ahlberg P. E., Trinajstic K., Mirone A. & P. Tafforeau. 2012. Three dimensional synchrotron virtual paleohistology: a new insight into the world of fossil bone microstructures. Microscopy and Microanalysis, 18-5: 1095-1105.

Sanchez S., Germain D., Ricqlès A. de, Abourachid A., Goussard F. & P. Tafforeau. 2010. Limb-bone histology of temnospondyls: implications for understanding the diversification of palaeoecologies and patterns of locomotion of Permo-Triassic tetrapods. Journal of Evolutionary Biology, 23-10: 2076-2090.

What can mineralised tissues tell us about the life history of early tetrapods?

Given that the transition from water to land entailed a major ecological shift, ultimately affecting every aspect of the animal's life, it seems very likely that it also involved significant changes in life history and reproductive strategy. We aim to test this hypothesis using skeletochronology, i.e. quantifying the growth lines in long bones. Preliminary results have demonstrated a great development plasticity of non-amniotic tetrapods to survive harsh environmental conditions during the Permian and Triassic (300-250 million years ago).

Figure 4: Geological map of the Saar-Nahe Basin in Germany presenting the locations where three palaeo-populations of the 300 million-year old tetrapod *Apateon* were excavated. Their different growth patterns suggest divergent life histories adapted to various environmental conditions.

Selected publications:

Sanchez S. & R. Schoch. 2013. Bone histology reveals a high environmental and metabolic plasticity as a successful evolutionary strategy in a long-lived homeostatic Triassic temnospondyl. Evolutionary Biology, 40-4: 627-647.

Sanchez S., Steyer J.S., Schoch R. & A. de Ricqlès. 2010. Palaeoecological and palaeoenvironmental influences revealed by long-bone palaeohistology: the example of the Permian branchiosaurid Apateon. In M. Vecoli, G. Clement and B. Meyer Berthaud, (eds.). The terrestrialization process: modelling complex interactions at the biosphere-geosphere Interface. The Geological Society, London, 339-1: 139-149.

Sanchez S., Klembara J., Castanet J. & J.S. Steyer. 2008. Salamander-like development in a seymouriamorph revealed by paleohistology. Biology Letters, 4: 411-414.