The preaxial development pattern of limbs in the early evolution of quadrupeds is related to the formation of five fingers/toes

In 1843, before Darwin's On the Origin of Species, British paleontologist and comparative anatomist Richard Owen explained in his masterpiece On the Nature of Limbs the idea
that the limb bones of amphibians, reptiles and mammals (collectively known as quadrupeds) originated from the fins of meat-finned fish.
Over the next 180 years, this idea appeared widely in textbooks and popular science books, and became a classic example
of homologous evolution in evolutionary biology.

With the rapid development of interdisciplinary disciplines in the past 30 years, the discovery of a large number of beautifully preserved "intermediate transition type" fossils has gradually unveiled the mystery in the evolutionary history from fish to quadrupeds, such as the loss of the dorsal fin and gill cap of fish, and the transformation
of the skull from flat left and right sides to dorsoventral to flat.
Most of the structures of quadrupeds' limbs are homologous to the thoracic/ventral fins of meat-finned fish, which is supported
by studies in morphology, developmental biology and molecular biology.
For example, the pectoral fin of a lungfish has roughly the same anatomy and articulation as a human arm, that is, it is articulated by a proximal long bone (humerus) and two distal long bones (ulna and radius), and then related to the proximal carpal bone
.
The distal pectoral fins (basal and membranous fins) of the lungfish and the distal carpal, metacarpal and phalanges of mammalian forelimbs have different morphologies and articular patterns, but have similar multiple gene expression patterns and regulatory mechanisms, such as being regulated
by the Hoxa13 and Hoxd13 genes in the early stages of embryonic development.
For more than a decade, there has been debate about whether carnivorous finned fish have fingers, and whether quadrupeds' fingers, metacarpals, and associated distal carpal bones represent an evolutionary novelty of
quadrupeds.

Unfortunately, paleontologists have been slow to find transitional morphological types
between the pectoral and ventral fins and the distal wrist/tarsal and fingers/toes and palm/tarsal bones of quadrupeds in the Devonian era (about 4.
2-360 million years ago).
Fossils of flesh-finned fish close to the common ancestor of tetrapods, such as Tiktaalik, have multiple basal bones and rays at the distal end of the pectoral fin; These basal bones and fins were lost during the evolution from fins to limbs and replaced
by wrist, palm, and phalanges.
On the other hand, the earliest quadrupeds found from the end of the Devonian period, such as Acanthostega, Ichthyostega and Tulerpeton, had 8, 7 and 6 fingers/toes on their limbs
, respectively.
These fossil records suggest a transition
from polydactyly/toed to five-fingered/toed in the early evolution of quadrupeds.

In addition, the study of embryonic development has opened a new chapter
in the understanding of the evolution from fins to limbs.
Previous studies have found that there is axial predominance in the early development process of the limb bones of the most primitive group of modern tetrapods, salamanders (a class of modern amphibians, represented by the Chinese giant salamander, commonly known as doll fish).
That is, the bone on the front side of the long axis (the side near the thumb) appears
before the bone on the back side.
For example, the toe formation order of the Mexican salamander is II-I-III-IV-V, with the tibia forming
preceding the fibula.
This developmental pattern is the opposite of postaxial dominance exhibited during the early development of limb bones in frogs and amniotic animals, such as frogs or turtles whose toes form in the order IV-V-III-II-I, and the tibia also
forms later than the fibula.
Modal differences in quadrupeds, especially non-amniotic animals, during early limb development are generally thought to exist later
in ossification.

Fig.
1 Macroscopic evolution of thoracic/ventral fins to quadruped limbs in meat-finned fish and comparison
of limb development polarity in modern tetrapods discovered by predecessors.

Over the past century, preaxial priority has been considered a peculiar, progressive developmental pattern for salamanders, while postaxial priority has represented the primitive developmental pattern
of quadrupeds.
In the last decade, researchers have found that preaxial preference also occurred during the ossification of the fingers and ulnar/radius of individual amphibian temnospondyls during the Permian period (2.
9-250 million years ago
).
However, the ossification pattern of the wrist/tarsal bone has not been studied
in the salamander and extinct primitive tetrapods due to the late ossification time and poor preservation of fossils.

Professor Gao Keqin's team at the Institute of Prehistoric Life and Environment, School of Earth and Space of Peking University has accumulated a large number of Mesozoic and modern salamander specimens and data
in the long-term research process.
Based on high-precision CT scanning and 3D reconstruction of 214 specimens, postdoctoral fellow Jia Jia and Professor Gao Keqin and domestic and foreign collaborators analyzed the ossification development pattern of the wrist/tarsal bone of 60 species in 6 families and 37 genera of axolotls
.
The study covers all modern and fossil lineages of the wrist/tarsal ossification of the axolotls, as well as a variety
of extinct early tetrapods, such as lepospondyls, detached vertebrae, and amniotic membranes.

The study found axial precedence to be present during distal wrist/tarsal ossification in multiple groups of quadrupeds, including most salamanders, 4 detached vertebrates (Archegosaurus, Amphibamus, Balanerpeton, Gerobatrachus), 1 protoconchopod (Sauropleura), 1 protoamniotic membrane (Diadectes), and 2 prototetrapods (Greererpeton, Proterogyrinus)
。 If the arm is taken as an example, the 1st and 2nd distal carpal bones are ossified before the 3rd and 4th distal carpal bones
.
Postaxial preference exists during ossification of the proximal carpal bones (e.
g.
, radial carpal, y-carpal, central carpal, ulnar carpal, and intercarpal bone) of quadrupeds, including all salamanders, and Australian lungfish, i.
e
.
, the radial carpal and y-carpal bones are always ossified last and have a significant delay.
At the same time, the axial priority also exists in the ossification process of the distal carpal bones of the two more advanced salamander groups (Salamanderidae and Alung newts), that is, the fourth distal carpal bone precedes the ossification
of the first and second distal carpal bones.
In addition, the ulna was found to be directly articulated with most of the basal bones and fins in flesh-finned fish close to the common ancestor of tetrapods, directly associated with the metacarpals and phalanges on the posterior side of the long axis in the quadruped basal stem taxa, and directly articulated with the distal carpal bone in the more advanced quadrupedian crest, and the distal carpal bone formed a one-to-one correspondence with the number of fingers
.
More interestingly, the work observed that the distal carpal bone was lost before the corresponding ulnar finger in early tetrapods, and later than the corresponding ulnar finger
in the salamander.

Fig.
2 Ossification patterns
of carpal bones and tarsal bones of Panhynobia, a primitive clade of salamanders.
The distal wrist/tarsal bone shows anterior axial priority, and the proximal wrist/tarsal bone shows posterior axial priority
.

Fig.
3 Ossification patterns
of carpal bones and tarsal bones of Salamandroidea, a progressive clade of salamanders.
Proximal wrist/tarsal of all genera and species and some of the non-lungulating newts (D, Aneides; F-H, Thorius) and salamander family (K, Lyciasalamandra; L,M, Notophthalmus; O, Paramesotriton) exhibits posterior axial preference in the distal carpal bone and the distal wrist/tarsal bone in the remaining genera exhibit axial priority
.

The study suggests that quadrupeds have different patterns of development inside their wrists/tarsal bones and should no longer be considered a whole
.
The distal wrist/tarsal bone has an independent developmental pattern and evolutionary history, and is an evolutionary new quality
of quadrupeds.
Nor should axial priority be thought of as a progressive, progressive pattern of development peculiar to salamanders or individual detached vertebrates, but rather a prevalent, primitive pattern
of limb bone development in early tetrapods.
The emergence of preaxial priority developmental patterns is not related to larval type (such as pond type, stream type) and life history strategy (such as abnormal development, larval persistence, etc.
), and is likely related to
the exercise stress constraints encountered during limb movement 。 Similar to the phenomenon that many structures that appear later in ontogeny are prone to be lost earlier in the evolutionary process, the axial priority displayed by the distal wrist/tarsal bone promotes the loss of the number of fingers/toes on the posterior side of the long axis of the limb bone, that is, the evolution of quadrupeds from early polydactyly/toe to five-finger/toe type, and also plays a stabilizing role
in the one-to-one relationship between the distal wrist/tarsal bone and the fingers/toes and metacarpals in the quadruped crown group.

Fig.
4 Limb bone development and evolution patterns of quadrupeds

Fig.
5 Early quadruped pentafinger/toe formation, origin and ossification polarity comparison of distal carpal bone and tarsal bone

The work was published in Science Advances under the title "Ossification patterns of the carpus and tarsus in salamanders and impacts of preaxial dominance on the fin-to-limb transition
.
" 。 Jia Jia (now a postdoctoral fellow at the University of Calgary, Canada) is the first author and corresponding author of the paper, and Professor Neil Shubin of the University of Chicago and Professor Gao Keqin of the School of Earth and Space are the co-corresponding authors
.
This work was supported
by the National Natural Science Foundation of China, the State Key Laboratory of Modern Paleontology and Stratigraphy, Nanjing Institute of Paleontology, Chinese Academy of Sciences, the Natural Science and Engineering Council of Canada Exploration Project, and the Pilot A Project of the Chinese Academy of Sciences.