Development and evolution of the tetrapod skull–neck boundary

The origin and evolution of the vertebrate skull have been topics of intense study for more than two centuries. Whereas early theories of skull origin, such as the influential vertebral theory, have been largely refuted with respect to the anterior (pre‐otic) region of the skull, the posterior (post‐otic) region is known to be derived from the anteriormost paraxial segments, i.e. the somites. Here we review the morphology and development of the occiput in both living and extinct tetrapods, taking into account revised knowledge of skull development by augmenting historical accounts with recent data. When occipital composition is evaluated relative to its position along the neural axis, and specifically to the hypoglossal nerve complex, much of the apparent interspecific variation in the location of the skull–neck boundary stabilizes in a phylogenetically informative way. Based on this criterion, three distinct conditions are identified in (i) frogs, (ii) salamanders and caecilians, and (iii) amniotes. The position of the posteriormost occipital segment relative to the hypoglossal nerve is key to understanding the evolution of the posterior limit of the skull. By using cranial foramina as osteological proxies of the hypoglossal nerve, a survey of fossil taxa reveals the amniote condition to be present at the base of Tetrapoda. This result challenges traditional theories of cranial evolution, which posit translocation of the occiput to a more posterior location in amniotes relative to lissamphibians (frogs, salamanders, caecilians), and instead supports the largely overlooked hypothesis that the reduced occiput in lissamphibians is secondarily derived. Recent advances in our understanding of the genetic basis of axial patterning and its regulation in amniotes support the hypothesis that the lissamphibian occipital form may have arisen as the product of a homeotic shift in segment fate from an amniote‐like condition.     

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.     

Refugia within refugia – patterns in endemism and genetic divergence are linked to Late Quaternary climate stability in the Iberian Peninsula

In Europe, southern peninsulas served as major refugia during Pleistocene cold periods. However, growing evidence has revealed complex patterns of glacial survival within these southern regions, with multiple glacial refugia within each larger refugial area. We investigated the extent to which patterns of endemism and phylogeographic are concordant across animal species in the Iberian Peninsula, one of the most important unglaciated areas in Europe during the Pleistocene, can be explained in terms of climatic stability. We found that historical climatic stability (notably climate velocity measures integrating macroclimatic shifts with local spatial topoclimate gradients) was often among the most important predictors of endemic species richness for different taxonomic groups using models that also incorporated measures of modern climate. Furthermore, for some taxonomic groups, climatic stability was also correlated with patterns of spatial concordance in interpopulation genetic divergence across multiple taxa, and private haplotypes were more frequently found in relatively stable areas. Overall, our results suggest that both endemism patterns and cross‐taxa concordant phylogeographic patterns across the Iberian Peninsula to some extent are linked to spatial variation in Late Quaternary climate stability, in agreement with the proposed ‘refugia‐within‐refugia’ scenario. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 13–28.     

Bystrow’s Paradox – gills,fossils, and the fish‐to‐tetrapod transition

Schoch, R.R. and Witzmann, F. 2011. Bystrow’s Paradox – gills, fossils, and the fish‐to‐tetrapod transition. —Acta Zoologica (Stockholm) 92 : 251–265. The issue of which breathing mechanism was used by the earliest tetrapods is still unsolved. Recent discoveries of stem tetrapods suggest the presence of internal gills and fish‐like underwater breathing. The same osteological features were used by Bystrow to infer a salamander‐like breathing through external gills in temnospondyl amphibians. This apparent contradiction – here called Bystrow’s Paradox – is resolved by reviewing the primary fossil evidence and the anatomy of the two gill types in extant taxa. Rather unexpectedly, we find that internal gills were present in a range of early crown tetrapods (temnospondyls), based on the anatomy of gill lamellae and location of branchial arteries on the ventral side of gill arch elements (ceratobranchials). Although it remains to be clarified which components are homologous in external and internal gills, both gill types are likely to have been present in Palaeozoic tetrapods – internal gills in aquatic adults of some taxa, and external gills in the larvae of these taxa and in larvae of numerous forms with terrestrial adults, which resorbed the external gills after the larval phase. Future developmental studies will hopefully clarify which mechanistic pathways are involved in gill formation and how these might have evolved.     

Comparable disparity in the appendicular skeleton across the fish–tetrapod transition,and the morphological gap between fish and tetrapod postcrania

Appendicular skeletal traits are used to quantify changes in morphological disparity and morphospace occupation across the fish–tetrapod transition and to explore the informativeness of different data partitions in phylogeny reconstruction. Anterior appendicular data yield trees that differ little from those built from the full character set, whilst posterior appendicular data result in considerable loss of phylogenetic resolution and tree branch rearrangements. Overall, there is a significant incongruence in the signals associated with pectoral and pelvic data. The appendicular skeletons of fish and tetrapods attain similar levels of morphological disparity (at least when data are rarefied at the maximum sample size for fish in our study) and occupy similarly sized regions of morphospace. However, fish appear more dispersed in morphospace than tetrapods do. All taxa show a heterogeneous distribution in morphospace, and there is a clear separation between fish and tetrapods despite the presence of several evolutionarily intermediate taxa.     

Functional analysis of chick heparan sulfate 6‐O‐sulfotransferases in limb bud development

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O‐sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O‐sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS‐6‐O‐sulfotransferases (HS6ST‐1 and HS6ST‐2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf‐8 and Fgf‐10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST‐2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf‐8 and Fgf‐10 expressions than that achieved with HS6ST‐1 RNAi. HS6ST‐1 RNAi and HS6ST‐2 RNAi caused a significant but distinct reduction in the levels of different 6‐O‐sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6‐O‐sulfation of HS play essential roles in chick limb bud development.     

Complex patterns of genetic and phenotypic divergence in an island bird and the consequences for delimiting conservation units

Substantial phenotypic and genetic variation is often found below the species level and this may be useful in quantifying biodiversity and predicting future diversification. However, relatively few studies have tested whether different aspects of intraspecific variation show congruent patterns across populations. Here, we quantify several aspects of divergence between 13 insular populations of an island endemic bird, the Vanuatu white-eye ( Zosterops flavifrons ). The components of divergence studied are mitochondrial DNA (mtDNA), nuclear DNA microsatellites and morphology. These different aspects of divergence present subtly different scenarios. For instance, an mtDNA phylogenetic tree reveals a potential cryptic species on the most southerly island in Vanuatu and considerable divergence between at least two other major phylogroups. Microsatellite loci suggest that population genetic divergence between insular populations, both between and within phylogroups, is substantial, a result that is consistent with a low level of interisland gene flow. Finally, most populations were found to be strongly morphologically divergent, but no single population was morphologically diagnosable from all others. Taken together, our results show that, although many measures of divergence are concordant in this system, the number of divergent units identified varies widely depending on the characters considered and approach used. A continuum of divergence and a degree of discordance between different characters are both to be expected under simple models of evolution, but they present problems in terms of delimiting conservation units.     

CO2‐metabolism in early tetrapods revisited: inferences from osteological correlates of gills,skin and lung ventilation in the fossil record

One of the most important physiological changes during the conquest of land by vertebrates was the increasing reliance on lung breathing, with the concomitant decrease in importance of gill breathing. The main problem involved here was to cope with the excessive accumulation of CO₂ in the body and to avoid respiratory acidosis. In the past, several often mutually contradicting hypotheses of CO₂‐elimination via skin, lungs and gills in early tetrapods have been proposed, based on theoretical physiological considerations and comparison with extant air‐breathing fishes and amphibians. This study proposes a revised scenario of CO₂‐elimination in early tetrapods based on fossil evidence, that is recently identified osteological correlates of gills, skin structure and mode of lung ventilation. In stem tetrapods of the Devonian and Carboniferous, O₂‐uptake via the lungs by buccal pumping was decoupled from CO₂‐release via internal gills, and the rather gas‐impermeable skin played a minor role in gaseous exchange. The two main lineages of crown‐group tetrapods, the amphibian and amniote lineage, used different strategies of CO₂‐elimination. As in stem tetrapods, O₂‐uptake and CO₂‐release remained always largely decoupled in temnospondyls, which ventilated their lungs via buccal pumping and relied mainly on their internal gills for CO₂‐release. Temnospondyls were not able to reduce their internal gills before their skin became more gas permeable and their body size was reduced, to shift from internal gills to the skin as the major site of CO₂‐elimination, a pattern that is retained in most lissamphibians. In contrast, internal gills were lost very early in stem amniote evolution. This was associated with the evolution of the more effective aspiration pump that allowed the elimination of the bulk of CO₂ via the lungs, leading to a coupled O₂‐uptake and CO₂‐loss in stem amniotes and later in amniotes.     

Lack of parallel genetic patterns underlying the repeated ecological divergence of beach and stream‐spawning kokanee salmon

Recent progress in methods for detecting adaptive population divergence in situ shows promise for elucidating the conditions under which selection acts to generate intraspecific diversity. Rapid ecological diversification is common in fishes; however, the role of phenotypic plasticity and adaptation to local environments is poorly understood. It is now possible to investigate genetic patterns to make inferences regarding phenotypic traits under selection and possible mechanisms underlying ecotype divergence, particularly where similar novel phenotypes have arisen in multiple independent populations. Here, we employed a bottom‐up approach to test for signatures of directional selection associated with divergence of beach‐ and stream‐spawning kokanee, the obligate freshwater form of sockeye salmon (Oncorhynchus nerka). Beach‐ and stream‐spawners co‐exist in many post‐glacial lakes and exhibit distinct reproductive behaviours, life‐history traits and spawning habitat preferences. Replicate ecotype pairs across five lakes in British Columbia, Canada were genotyped at 57 expressed sequence tag‐linked and anonymous microsatellite loci identified in a previous genome scan. Fifteen loci exhibited signatures of directional selection (high F_ST outliers), four of which were identified in multiple lakes. However, the lack of parallel genetic patterns across all lakes may be a result of: 1) an inability to detect loci truly under selection; 2) alternative genetic pathways underlying ecotype divergence in this system; and/or 3) phenotypic plasticity playing a formative role in driving kokanee spawning habitat differences. Gene annotations for detected outliers suggest pathogen resistance and energy metabolism as potential mechanisms contributing to the divergence of beach‐ and stream‐spawning kokanee, but further study is required.     

Sox9‐expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons

Sox9 expression defines cell progenitors in a variety of tissues during mouse embryogenesis. To establish a genetic tool for cell‐lineage tracing and gene‐function analysis, we generated mice in which the CreERT2 gene was targeted to the endogenous mouse Sox9 locus. In Sox9^CreERT2/+;R26R embryos, tamoxifen activated Cre recombinase exclusively in Sox9‐expressing tissues. To determine the suitability of this mouse line for developmental stage‐specific gene recombination, we investigated the cellular origins of the cruciate ligaments of the knee joint and the limb tendons, in which precursor cells have not been defined. The cells in these tissues were labeled after tamoxifen treatment before or at the stage of chondrogenic mesenchymal condensation, indicating that ligament and tendon cells originated from Sox9‐expressing cells and that cell fate determination occurred at mesenchymal condensation. This mouse line is a valuable tool for the temporal genetic tracing of the progeny of, and inducible gene modification in Sox9‐expressing cells. genesis 48:635–644, 2010. © 2010 Wiley‐Liss, Inc.     

Generation of Shox2‐Cre allele for tissue specific manipulation of genes in the developing heart,palate, and limb

Shox2 is expressed in several developing organs in a tissue specific manner in both mice and humans, including the heart, palate, limb, and nervous system. To better understand the spatial and temporal expression patterns of Shox2 and to systematically dissect the genetic cascade regulated by Shox2, we created Shox2‐LacZ and Shox2‐Cre knock‐in mouse lines. We show that the Shox2‐LacZ allele expresses beta‐galactosidase reporter gene in a fashion that recapitulates the endogenous Shox2 expression pattern in developing organs, including the sinoatrial node (SAN), the anterior portion of the palate, and the proximal region of the limb bud. Conditional deletion of Shox2 in mice carrying the Shox2‐Cre allele yielded SAN phenotypes that resemble conventional Shox2 knockout mice. Our results indicate that the Shox2‐Cre allele offer a useful tool for tissue specific manipulation of genes in a number of developing organs, particularly in the developing SAN. genesis 51:515–522. © 2013 Wiley Periodicals, Inc.     

Genetic divergence and migration patterns in a North American passerine bird: implications for evolution and conservation

Like many other migratory birds, the black-throated blue warbler (Dendroica caerulescens) shows pronounced differences in migratory behaviour and other traits between populations: birds in the southern part of the breeding range have darker plumage and migrate to the eastern Caribbean during the winter, whereas those in the north have lighter plumage and migrate to the western Caribbean. We examined the phylogeography of this species, using samples collected from northern and southern populations, to determine whether differentiation between these populations dates to the Pleistocene or earlier, or whether differences in plumage and migratory behaviour have arisen more recently. We analysed variation at 369 bp of the mitochondrial control region domain I and also at seven nuclear microsatellites. Analyses revealed considerable genetic variation, but the vast majority of this variation was found within rather than between populations, and there was little differentiation between northern and southern populations. Phylogeographic analyses revealed a very shallow phylogenetic tree, a star-like haplotype network, and a unimodal mismatch distribution, all indicative of a recent range expansion from a single refugium. Coalescent modelling approaches also indicated a recent common ancestor for the entire group of birds analysed, no split between northern and southern populations, and high levels of gene flow. These results show that Pleistocene or earlier events have played little role in creating differences between northern and southern populations, suggesting that migratory and other differences between populations have arisen very recently. The implications of these results for the evolution of migration and defining taxonomic groups for conservation efforts are discussed.     

Msx1‐2 immunolocalization in the regenerating tail of a lizard but not in the scarring limb suggests its involvement in the process of regeneration

The immunolocalization of the muscle segmental homoeobox protein Msx1‐2 of 27–34 kDa in the regenerating tail blastema of a lizard shows prevalent localization in the apical ependyma of the regenerating spinal cord and less intense labelling in the wound epidermis, in the apical epidermal peg (AEP), and in the regenerating segmental muscles. The AEP is a micro‐region of the regenerating epidermis located at the tail tip of the blastema, likely corresponding to the AEC of the amphibian blastema. No immunolabelling is present in the wound epidermis and scarring blastema of the limb at 18–21 days of regeneration, except for sparse repairing muscles. The presence of a proximal–distal gradient of Msx1‐2 protein, generated from the apical ependyma, is suggested by the intensity of immunolabelling. The AEP and the ependyma are believed to induce and maintain tail regeneration, and this study suggests that Msx1‐2 proteins are components of the signalling system that maintains active growth of the tail blastema. The lack of activation and production of Msx1‐2 protein in the limb are likely due to the intense inflammatory reaction following amputation. This study confirms that, like during regeneration in fishes and amphibians, also the blastema of lizards utilizes common signalling pathways for maintaining regeneration.