Selection on Phalanx Development in the Evolution of the Bird Wing.

The frameshift hypothesis is a widely accepted model of bird wing evolution. This hypothesis postulates a shift in positional values, or molecular-developmental identity, that caused a change in digit phenotype. The hypothesis synthesized developmental and paleontological data on wing digit homology. The "most anterior digit" (MAD) hypothesis presents an alternative view based on changes in transcriptional regulation in the limb. The molecular evidence for both hypotheses is that the MAD expresses Hoxd13 but not Hoxd11 and Hoxd12. This digit I "signature" is thought to characterize all amniotes. Here, we studied Hoxd expression patterns in a phylogenetic sample of 18 amniotes. Instead of a conserved molecular signature in digit I, we find wide variation of Hoxd11, Hoxd12, and Hoxd13 expression in digit I. Patterns of apoptosis, and Sox9 expression, a marker of the phalanx-forming region, suggest that phalanges were lost from wing digit IV because of early arrest of the phalanx-forming region followed by cell death. Finally, we show that multiple amniote lineages lost phalanges with no frameshift. Our findings suggest that the bird wing evolved by targeted loss of phalanges under selection. Consistent with our view, some recent phylogenies based on dinosaur fossils eliminate the need to postulate a frameshift in the first place. We suggest that the phenotype of the Archaeopteryx lithographica wing is also consistent with phalanx loss. More broadly, our results support a gradualist model of evolution based on tinkering with developmental gene expression.

Noncanonical Hox, Etv4, and Gli3 gene activities give insight into unique limb patterning in salamanders.

Limb development in salamanders is unique among tetrapods in significant ways. Not only can salamanders regenerate lost limbs repeatedly and throughout their lives, but also the preaxial zeugopodial element and digits form before the postaxial ones and, hence, with a reversed polarity compared to all other tetrapods. Moreover, in salamanders with free-swimming larval stages, as exemplified by the axolotl (Ambystoma mexicanum), each digit buds independently, instead of undergoing a paddle stage. Here, we report gene expression patterns of Hoxa and d clusters, and other crucial transcription factors during axolotl limb development. During early phases of limb development, expression patterns are mostly similar to those reported for amniotes and frogs. Likewise, Hoxd and Shh regulatory landscapes are largely conserved. However, during late digit-budding phases, remarkable differences are present: (i) the Hoxd13 expression domain excludes developing digits I and IV, (ii) we expand upon previous observation that Hoxa11 expression, which traditionally marks the zeugopodium, extends distally into the developing digits, and (iii) Gli3 and Etv4 show prolonged expression in developing digits. Our findings identify derived patterns in the expression of key transcription factors during late phases of salamander limb development, and provide the basis for a better understanding of the unique patterning of salamander limbs.

Hox gene expression in the specialized limbs of the Iberian mole (Talpa occidentalis).

Fossorial talpid moles use their limbs predominantly for digging, which explains their highly specialized anatomy. The humerus is particularly short and dorsoventrally rotated, with broadened distal and proximal parts where muscles attach and which facilitate powerful abductive movements. The radius and ulna are exceptionally robust and short. The ulna has an expanded olecranon process. The femur is generalized, but the fused tibia-fibula complex is short and robust. To understand the developmental bases of these specializations, we studied expression patterns of four 5' Hox genes in the fossorial Iberian mole (Talpa occidentalis). These genes are known to play major roles in patterning the developing limb skeleton in the mouse, with which comparisons were made (Mus musculus, C57BL/6Jico strain). We find that HoxA9 expression is spatially expanded in the developing stylopodial area in the mole forelimb, compared to the less specialized mouse forelimb and mole hind limb. HoxD9 expression does not extend into the thoracic body wall in the mole forelimb in contrast to the mouse, and is also reduced in the presumptive zeugopodium in mole forelimb, compared to mouse. Expression of HoxD11 is upregulated in the mole in the postaxial area of the hind limb zeugopod, compared to the mouse. On the other hand, HoxD13 is downregulated in the postaxial zeugopodial area in the forelimb of the mole, compared to the mouse. The differences in the expression patterns of these 5' Hox genes between Talpa and Mus are an indication of the developmental changes going hand in hand with anatomical digging adaptations in the mole adult.

The molecular origin and evolution of dim-light vision in mammals.

The nocturnal origin of mammals is a longstanding hypothesis that is considered instrumental for the evolution of endothermy, a potential key innovation in this successful clade. This hypothesis is primarily based on indirect anatomical inference from fossils. Here, we reconstruct the evolutionary history of rhodopsin--the vertebrate visual pigment mediating the first step in phototransduction at low-light levels--via codon-based model tests for selection, combined with gene resurrection methods that allow for the study of ancient proteins. Rhodopsin coding sequences were reconstructed for three key nodes: Amniota, Mammalia, and Theria. When expressed in vitro, all sequences generated stable visual pigments with λMAX values similar to the well-studied bovine rhodopsin. Retinal release rates of mammalian and therian ancestral rhodopsins, measured via fluorescence spectroscopy, were significantly slower than those of the amniote ancestor, indicating altered molecular function possibly related to nocturnality. Positive selection along the therian branch suggests adaptive evolution in rhodopsin concurrent with therian ecological diversification events during the Mesozoic that allowed for an exploration of the environment at varying light levels.

Deep-time evolution of regeneration and preaxial polarity in tetrapod limb development.

Among extant tetrapods, salamanders are unique in showing a reversed preaxial polarity in patterning of the skeletal elements of the limbs, and in displaying the highest capacity for regeneration, including full limb and tail regeneration. These features are particularly striking as tetrapod limb development has otherwise been shown to be a highly conserved process. It remains elusive whether the capacity to regenerate limbs in salamanders is mechanistically and evolutionarily linked to the aberrant pattern of limb development; both are features classically regarded as unique to urodeles. New molecular data suggest that salamander-specific orphan genes play a central role in limb regeneration and may also be involved in the preaxial patterning during limb development. Here we show that preaxial polarity in limb development was present in various groups of temnospondyl amphibians of the Carboniferous and Permian periods, including the dissorophoids Apateon and Micromelerpeton, as well as the stereospondylomorph Sclerocephalus. Limb regeneration has also been reported in Micromelerpeton, demonstrating that both features were already present together in antecedents of modern salamanders 290 million years ago. Furthermore, data from lepospondyl 'microsaurs' on the amniote stem indicate that these taxa may have shown some capacity for limb regeneration and were capable of tail regeneration, including re-patterning of the caudal vertebral column that is otherwise only seen in salamander tail regeneration. The data from fossils suggest that salamander-like regeneration is an ancient feature of tetrapods that was subsequently lost at least once in the lineage leading to amniotes. Salamanders are the only modern tetrapods that retained regenerative capacities as well as preaxial polarity in limb development.

Early evolution of limb regeneration in tetrapods: evidence from a 300-million-year-old amphibian.

Salamanders are the only tetrapods capable of fully regenerating their limbs throughout their entire lives. Much data on the underlying molecular mechanisms of limb regeneration have been gathered in recent years allowing for new comparative studies between salamanders and other tetrapods that lack this unique regenerative potential. By contrast, the evolution of animal regeneration just recently shifted back into focus, despite being highly relevant for research designs aiming to unravel the factors allowing for limb regeneration. We show that the 300-million-year-old temnospondyl amphibian Micromelerpeton, a distant relative of modern amphibians, was already capable of regenerating its limbs. A number of exceptionally well-preserved specimens from fossil deposits show a unique pattern and combination of abnormalities in their limbs that is distinctive of irregular regenerative activity in modern salamanders and does not occur as variants of normal limb development. This demonstrates that the capacity to regenerate limbs is not a derived feature of modern salamanders, but may be an ancient feature of non-amniote tetrapods and possibly even shared by all bony fish. The finding provides a new framework for understanding the evolution of regenerative capacity of paired appendages in vertebrates in the search for conserved versus derived molecular mechanisms of limb regeneration.

Evolution of bone compactness in extant and extinct moles (Talpidae): exploring humeral microstructure in small fossorial mammals.

BACKGROUND: Talpids include forms with different degree of fossoriality, with major specializations in the humerus in the case of the fully fossorial moles. We studied the humeral microanatomy of eleven extant and eight extinct talpid taxa of different lifestyles and of two non-fossorial outgroups and examined the effects of size and phylogeny. We tested the hypothesis that bone microanatomy is different in highly derived humeri of fossorial taxa than in terrestrial and semi-aquatic ones, likely due to special mechanical strains to which they are exposed to during digging. This study is the first comprehensive examination of histological parameters in an ecologically diverse and small-sized mammalian clade. RESULTS: No pattern of global bone compactness was found in the humeri of talpids that could be related to biomechanical specialization, phylogeny or size. The transition zone from the medullary cavity to the cortical compacta was larger and the ellipse ratio smaller in fossorial talpids than in non-fossorial talpids. No differences were detected between the two distantly related fossorial clades, Talpini and Scalopini. CONCLUSIONS: At this small size, the overall morphology of the humerus plays a predominant role in absorbing the load, and microanatomical features such as an increase in bone compactness are less important, perhaps due to insufficient gravitational effects. The ellipse ratio of bone compactness shows relatively high intraspecific variation, and therefore predictions from this ratio based on single specimens are invalid.

Transcriptional heterochrony in talpid mole autopods.

BACKGROUND: Talpid moles show many specializations in their adult skeleton linked to fossoriality, including enlarged hands when compared to the feet. Heterochrony in developmental mechanisms is hypothesized to account for morphological evolution in skeletal elements. METHODS: The temporal and spatial distribution of SOX9 expression, which is an early marker of chondrification, is analyzed in autopods of the fossorial Iberian mole Talpa occidentalis, as well as in shrew (Cryptotis parva) and mouse (Mus musculus) for comparison. RESULTS AND DISCUSSION: SOX9 expression is advanced in the forelimb compared to the hind limb in the talpid mole. In contrast, in the shrew and the mouse, which do not show fossorial specializations in their autopods, it is synchronous. We provide evidence that transcriptional heterochrony affects the development of talpid autopods, an example of developmental penetrance. We discuss our data in the light of earlier reported pattern heterochrony and later morphological variation in talpid limbs. CONCLUSION: Transcriptional heterochrony in SOX9 expression is found in talpid autopods, which is likely to account for pattern heterochrony in chondral limb development as well as size variation in adult fore- and hind limbs.

Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal.

Monotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λ(max) of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min-1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min-1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.