Final Report Summary - TETRAPODS RISING (Tetrapods Rising: Linking changes in mandibular form with function across the fish-tetrapod transition)
The invasion of the land by vertebrates was one of the great transitions in the history of life. Numerous fossils document the metamorphosis from aquatic, lobe-finned fishes to terrestrial tetrapods. Previous studies have focused on the evolution of the limbs, axial skeleton and cranium in early tetrapods; in contrast, the lower jaw has received little attention. Yet stepwise changes in lower jaw shape during the water-land transition have been qualitatively linked to changes in feeding behaviour and/or environment. The aim of this project is to understand how changes in the structure of the lower jaw along the tetrapod lineage and during a key evolutionary transition (water to land) are linked to mechanical behaviour and ecology.
Work carried out to achieve project objectives: From 2012-2014, Dr Porro carried out computed tomographic (CT) scans of more than 15 fossil skulls, representing nine early tetrapod species. Additionally, the heads of two extant fish - the European eel (Anguilla anguilla) and pike (Esox lucius) - were CT-scanned before and after staining with iodine potassium iodide (IKI) in order to visualize both hard and soft tissues. Visualization software (Avizo) was used to digitally strip fossil specimens of matrix (and separate bone and muscle tissue in extant species) and separate individual bones and teeth from each other. In the case of damaged or deformed fossil specimens, novel techniques were developed to reconstruct the skull in 3D (Figure 1).
In order to accurately model feeding behaviour and validate models, data was needed from living taxa. In vivo feeding behavior in Esox and Anguilla was recorded using high-speed video and bite forces were measured using force transducers. Nanoindentation was used to obtain bone material properties from the lower jaws of Esox and Anguilla and information from contrast-enhanced CT-scans revealed jaw muscle architecture in unprecedented detail (Figure 2). Together with 3D surface models (see above) this information was used to produced high-resolution, 3D finite element (FE) models of the lower jaws in the software Strand7. Models were scaled (using Anguilla as the reference taxon) to produce two sets of models - Set 1 models were scaled to equal surface area, Set 2 models were scaled to equal volume. Models were assigned appropriate material properties, constrained and loaded with forces exerted by jaw muscles. Data from these FE models, including deformation, strain and stress under loads, were used to understand and quantify the mechanical behaviour of the lower jaw under feeding loads (Figure 3).
Main results, conclusions and impacts: CT-scanning and digital preparation of fossil specimens revealed details previously obscured by matrix or superficial bones, providing a wealth of new anatomical information on skull anatomy in early tetrapods. Novel methods to retrodeform the skull resulted in 3D reconstructions that differ in important ways from previous efforts. Dr Porro has produced a complete anatomical description and 3D reconstruction pf the skull of the iconic early tetrapod Acanthostega (accepted for publication in PLoS ONE) and two additional descriptive papers on the lower jaws of Eusthenopteron and Crassigyrinus (to be submitted shortly).
In vivo feeding experiments with Esox revealed that this taxon captures and ingests prey using suction, contrary to its widespread reputation as a biter. Contrast-enhanced CT scanning of the heads of Esox and Anguilla using IKI was highly successful and revealed cranial musculature in unprecedented detail; this data has become the basis for an MSc project (supervised by Dr Porro and the host PI Professor Rayfield) and will be submitted for publication in the coming months. Nanoindentation permitted measurements of the elastic moduli of the lower jaws of Esox and Anguilla; as material properties data for fish skull bone are practically non-existent in the literature, these data represent an important contribution to the literature. Our findings show that lower jaw bone in Anguilla, which captures and processes prey by biting, is stiffer and more anisotropic than that of Esox, a suction feeder.
Output from FE models of the lower jaws of four early tetrapods, two extant fish and Alligator were compared to address questions on the link between changes in lower jaw morphology and mechanical behaviour. Results from finite element analysis (FEA) reveal that deformation regimes in fish, early tetrapod and Alligator lower jaws under applied feeding loads were similar; however, the magnitude of deformation was higher in the extant fish than in the fossil tetrapods while deformation was lowest in Alligator and the early tetrapodomorph fish Eusthenopteron. Coupled with similar patterns in overall stress magnitude (highest in fish, lowest in Alligator) these results support our hypothesis that changes in mandibular morphology during tetrapod evolution (such as closure of the Meckelian fenestra, decreased number of bones in the lower jaw and the development of overlapping sutures) resulted in a stronger lower jaw. Stress was more evenly distributed in the lower jaws of tetrapods than in fish, and generally more homogeneous in more derived tetrapods (including Alligator) than in basal forms. Total strain energy generally decreased along the lineage, indicating that less applied (=muscle) force is being expended to deform the model and suggesting a stiffer, more efficient structure (the only exception to this pattern was Acanthostega, which exhibited higher strain energy than the more basal Eusthenopteron). Together, results from stress distribution and total strain energy support our hypothesis that the lower jaw became better adapted to biting during the evolution of tetrapods, possibly due to release from the need to be hydrodynamically streamlined as tetrapods moved onto land.
Unexpected findings include the occurrence of the highest stress (during the application of feeding loads) on the biting side of the lower jaw in fish; however, this area of high stress shifts to the symphysis in the early tetrapodomorph Eusthenopteron and to the non-biting (balancing) side in later tetrapods, including Alligator. We propose that the most likely explanation for this shift in the location of highest stress during biting is more efficient transfer of force across the mandibular symphysis due to increasingly complex symphyseal morphology. Furthermore, we found that the lower jaw of Eusthenopteron exhibits lower stress and lower total strain energy than that of Acanthostega. We suggest that the loss of Meckelian bone coupled with delayed closure of the Meckelian fenestra in Acanthostega resulted in a weaker lower jaw in this taxon. Results from finite element analysis serve as the basis for a single high-impact paper currently in preparation to be targeted at the journals Nature or Science.
Other impacts of the project: Our work has set a benchmark for performing FEA in a broader evolutionary context than previously attempted and established methods for 3D reconstruction of fossil skulls. We have gained a new understanding of the morphological and functional evolution of the tetrapod lower jaw during the water-land transition and generated large, novel data sets that will benefit future researchers. In addition to the accepted, submitted and proposed publications previously detailed, Dr Porro has presented results from this project at major international conferences, including: the International Congress of Vertebrate Morphology in Barcelona, Spain (July 2013), Annual Meetings of the Society of Vertebrate Paleontology (Los Angeles, USA in November 2013 and Berlin, Germany in November 2014) and the International Palaeontological Congress in Mendoza, Argentina (September 2014) as well as invited talks at several academic institutions. Dr. Porro has been involved in public engagement activities in which research and results were presented through major outreach events and informal presentations to interested amateur groups. 3D PDFs of several taxa from this study are now used in teaching courses at the University of Cambridge and the University of Bristol. Dr Porro has contributed to websites including the TWeed project page (http://tetrapods.org/) and Animal Bytes (http://animalbytescambridge.wordpress.com/).
Work carried out to achieve project objectives: From 2012-2014, Dr Porro carried out computed tomographic (CT) scans of more than 15 fossil skulls, representing nine early tetrapod species. Additionally, the heads of two extant fish - the European eel (Anguilla anguilla) and pike (Esox lucius) - were CT-scanned before and after staining with iodine potassium iodide (IKI) in order to visualize both hard and soft tissues. Visualization software (Avizo) was used to digitally strip fossil specimens of matrix (and separate bone and muscle tissue in extant species) and separate individual bones and teeth from each other. In the case of damaged or deformed fossil specimens, novel techniques were developed to reconstruct the skull in 3D (Figure 1).
In order to accurately model feeding behaviour and validate models, data was needed from living taxa. In vivo feeding behavior in Esox and Anguilla was recorded using high-speed video and bite forces were measured using force transducers. Nanoindentation was used to obtain bone material properties from the lower jaws of Esox and Anguilla and information from contrast-enhanced CT-scans revealed jaw muscle architecture in unprecedented detail (Figure 2). Together with 3D surface models (see above) this information was used to produced high-resolution, 3D finite element (FE) models of the lower jaws in the software Strand7. Models were scaled (using Anguilla as the reference taxon) to produce two sets of models - Set 1 models were scaled to equal surface area, Set 2 models were scaled to equal volume. Models were assigned appropriate material properties, constrained and loaded with forces exerted by jaw muscles. Data from these FE models, including deformation, strain and stress under loads, were used to understand and quantify the mechanical behaviour of the lower jaw under feeding loads (Figure 3).
Main results, conclusions and impacts: CT-scanning and digital preparation of fossil specimens revealed details previously obscured by matrix or superficial bones, providing a wealth of new anatomical information on skull anatomy in early tetrapods. Novel methods to retrodeform the skull resulted in 3D reconstructions that differ in important ways from previous efforts. Dr Porro has produced a complete anatomical description and 3D reconstruction pf the skull of the iconic early tetrapod Acanthostega (accepted for publication in PLoS ONE) and two additional descriptive papers on the lower jaws of Eusthenopteron and Crassigyrinus (to be submitted shortly).
In vivo feeding experiments with Esox revealed that this taxon captures and ingests prey using suction, contrary to its widespread reputation as a biter. Contrast-enhanced CT scanning of the heads of Esox and Anguilla using IKI was highly successful and revealed cranial musculature in unprecedented detail; this data has become the basis for an MSc project (supervised by Dr Porro and the host PI Professor Rayfield) and will be submitted for publication in the coming months. Nanoindentation permitted measurements of the elastic moduli of the lower jaws of Esox and Anguilla; as material properties data for fish skull bone are practically non-existent in the literature, these data represent an important contribution to the literature. Our findings show that lower jaw bone in Anguilla, which captures and processes prey by biting, is stiffer and more anisotropic than that of Esox, a suction feeder.
Output from FE models of the lower jaws of four early tetrapods, two extant fish and Alligator were compared to address questions on the link between changes in lower jaw morphology and mechanical behaviour. Results from finite element analysis (FEA) reveal that deformation regimes in fish, early tetrapod and Alligator lower jaws under applied feeding loads were similar; however, the magnitude of deformation was higher in the extant fish than in the fossil tetrapods while deformation was lowest in Alligator and the early tetrapodomorph fish Eusthenopteron. Coupled with similar patterns in overall stress magnitude (highest in fish, lowest in Alligator) these results support our hypothesis that changes in mandibular morphology during tetrapod evolution (such as closure of the Meckelian fenestra, decreased number of bones in the lower jaw and the development of overlapping sutures) resulted in a stronger lower jaw. Stress was more evenly distributed in the lower jaws of tetrapods than in fish, and generally more homogeneous in more derived tetrapods (including Alligator) than in basal forms. Total strain energy generally decreased along the lineage, indicating that less applied (=muscle) force is being expended to deform the model and suggesting a stiffer, more efficient structure (the only exception to this pattern was Acanthostega, which exhibited higher strain energy than the more basal Eusthenopteron). Together, results from stress distribution and total strain energy support our hypothesis that the lower jaw became better adapted to biting during the evolution of tetrapods, possibly due to release from the need to be hydrodynamically streamlined as tetrapods moved onto land.
Unexpected findings include the occurrence of the highest stress (during the application of feeding loads) on the biting side of the lower jaw in fish; however, this area of high stress shifts to the symphysis in the early tetrapodomorph Eusthenopteron and to the non-biting (balancing) side in later tetrapods, including Alligator. We propose that the most likely explanation for this shift in the location of highest stress during biting is more efficient transfer of force across the mandibular symphysis due to increasingly complex symphyseal morphology. Furthermore, we found that the lower jaw of Eusthenopteron exhibits lower stress and lower total strain energy than that of Acanthostega. We suggest that the loss of Meckelian bone coupled with delayed closure of the Meckelian fenestra in Acanthostega resulted in a weaker lower jaw in this taxon. Results from finite element analysis serve as the basis for a single high-impact paper currently in preparation to be targeted at the journals Nature or Science.
Other impacts of the project: Our work has set a benchmark for performing FEA in a broader evolutionary context than previously attempted and established methods for 3D reconstruction of fossil skulls. We have gained a new understanding of the morphological and functional evolution of the tetrapod lower jaw during the water-land transition and generated large, novel data sets that will benefit future researchers. In addition to the accepted, submitted and proposed publications previously detailed, Dr Porro has presented results from this project at major international conferences, including: the International Congress of Vertebrate Morphology in Barcelona, Spain (July 2013), Annual Meetings of the Society of Vertebrate Paleontology (Los Angeles, USA in November 2013 and Berlin, Germany in November 2014) and the International Palaeontological Congress in Mendoza, Argentina (September 2014) as well as invited talks at several academic institutions. Dr. Porro has been involved in public engagement activities in which research and results were presented through major outreach events and informal presentations to interested amateur groups. 3D PDFs of several taxa from this study are now used in teaching courses at the University of Cambridge and the University of Bristol. Dr Porro has contributed to websites including the TWeed project page (http://tetrapods.org/) and Animal Bytes (http://animalbytescambridge.wordpress.com/).