Terrestriality constrains salamander limb diversification: Implications for the evolution of pentadactyly

Patterns of phenotypic evolution can abruptly shift as species move between adaptive zones. Extant salamanders display three distinct life cycle strategies that range from aquatic to terrestrial (biphasic), to fully aquatic (paedomorphic) and to fully terrestrial (direct development). Life cycle variation is associated with changes in body form such as loss of digits, limb reduction or body elongation. However, the relationships among these traits and life cycle strategy remain unresolved. Here, we use a Bayesian modelling approach to test whether life cycle transitions by salamanders have influenced rates, optima and integration of primary locomotory structures (limbs and trunk). We show that paedomorphic salamanders have elevated rates of limb evolution with optima shifted towards smaller size and fewer digits compared to all other salamanders. Rate of hindlimb digit evolution is shown to decrease in a gradient as life cycles become more terrestrial. Paedomorphs have a higher correlation between hindlimb digit loss and increases in vertebral number, as well as reduced correlations between limb lengths. Our results support the idea that terrestrial plantigrade locomotion constrains limb evolution and, when lifted, leads to higher rates of trait diversification and shifts in optima and integration. The basic tetrapod body form of most salamanders and the independent losses of terrestrial life stages provide an important framework for understanding the evolutionary and developmental mechanisms behind major shifts in ecological zones as seen among early tetrapods during their transition from water to land.     

Explanation for naturally occurring supernumerary limbs in amphibians

The occasional occurrence of high frequencies of limb abnormalities, including extra limbs, in natural populations of amphibians has long been a puzzle. In this paper we report the discovery of a population in which such limb abnormalities appear to be caused by a parasitic flatworm (trematode) that uses amphibians as intermediate hosts. The cercarial larval stage of the trematode attacks amphibians, penetrating the skin to form cysts (metacercariae). The cysts are preferentially localized in the cloacal region, including the developing hind limb regions in larvae of both frogs (Hyla regilla) and salamanders (Ambystoma macrodactylum). A wide range of limb abnormalities are seen, including duplicated limb structures ranging from extra digits to several extra whole limbs. We hypothesize that these limb abnormalities result from localized regulatory responses of developing and regenerating limb tissues to mechanical disruption caused by the trematode cysts. We have tested this idea by implanting inert resin beads into developing limb buds of frogs and salamanders. Since this treatment can cause supernumerary limb structures, our hypothesis is sufficient to explain the naturally occurring extra limbs.     

Biological controls upon the physical taphonomy of exceptionally preserved salamanders from the Miocene of Rubielos de Mora,northeast Spain

McNamara, M.E., Orr, P.J., Manzocchi, T., Alcalá, L., Anadón, P. & Peñalver, E. 2011: Biological controls upon the physical taphonomy of exceptionally preserved salamanders from the Miocene of Rubielos de Mora, northeast Spain. Lethaia, Vol. 45, pp. 210–226. The middle Miocene Rubielos de Mora Konservat‐Lagerstätte of northeast Spain is hosted within profundal, finely laminated, lacustrine mudstones. The diverse biota includes abundant salamanders. Most individuals died during separate episodes and sank rapidly postmortem. Specimens are typically preserved in dorso‐ventral aspect, the most hydrodynamically stable orientation. The near‐cylindrical morphology of the body, however, allowed some carcasses to settle in or subsequently re‐orientate into, lateral orientations. Loss of skeletal elements (i.e. reduced completeness) reflects their location within the body and followed a distal to proximal trend. Two stages are identified: initial loss of a small number of phalanges, followed by loss of more proximal limb bones plus additional phalanges. Disarticulation is more complex: it occurred via several mechanisms (notably, abdominal rupture and re‐orientation of part of the body and limbs during decay) and shows no consistent pattern among specimens. The physical taphonomy of the salamanders is controlled predominantly by intrinsic biological factors, i.e. the geometry of the body and of individual skeletal elements, the orientation, inherent strength and location of specific joints and the extent to which soft tissues, particularly the skin, persist during decay. These biological factors probably control patterns of physical taphonomy of other fossil tetrapods with a similar skeletal configuration. □Articulation, completeness, Konservat‐Lagerstätten, orientation, quantitative taphonomy, salamanders.     

Early limb patterning in the direct‐developing salamander Plethodon cinereus revealed by sox9 and col2a1

Direct‐developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct‐developing frogs, but little attention has been given to direct‐developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct‐developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior‐to‐posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote‐like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct‐developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct‐developing salamander species.     

产前超声诊断及漏诊胎儿肢体畸形分析

目的:探讨胎儿肢体畸形超声特征及诊断价值。方法:采用连续顺序追踪法对66342例妊娠12-40周孕妇行胎儿四肢畸形筛查。将产前超声诊断结果与引产或产后结果进行对比分析。结果:发生肢体畸形271例,发生率为0.41%(271/66342),包括四肢短小5例,桡骨发育不全1例,缺肢畸形5例,足内翻17例,手掌畸形3例,指趾畸形222例及骨骼多发畸形18例。其中产前诊断胎儿肢体畸形49例;漏诊222例,包括:足内翻3例、指趾畸形218例、多发骨骼畸形1例。胎儿肢体畸形的出现率和产前检出率分别为:四肢短小1.84%(5/271)、100%(5/5);桡骨发育不全0.36%(1/271)、100%(1/1);缺肢畸形1.84%(5/271)、100%(5/5);足内翻6.27%(17/271)、82.35%(14/17);手掌畸形1.10%(3/271)、100%(3/3);指趾畸形81.91%(222/217)、1.8%(4/222);多发骨骼畸形6.64%(18/271)、94.44%(17/18)。结论:超声对胎儿手掌、脚掌部位以上畸形的检出率较高。指趾畸形出现率最高,但检出率最低。     

产前超声诊断及漏诊胎儿肢体畸形分析

目的：探讨胎儿肢体畸形超声特征及诊断价值。方法：采用连续顺序追踪法对66342 例妊娠12-40 周孕妇行胎儿四肢畸形筛查。将产前超声诊断结果与引产或产后结果进行对比分析。结果：发生肢体畸形271 例,发生率为0.41 %（271/66342）,包括四肢短小5 例,桡骨发育不全1 例,缺肢畸形5 例,足内翻17 例,手掌畸形3 例,指趾畸形222 例及骨骼多发畸形18 例。其中产前诊断胎儿肢体畸形49 例;漏诊222 例,包括：足内翻3 例、指趾畸形218 例、多发骨骼畸形1 例。胎儿肢体畸形的出现率和产前检出率分别为：四肢短小1.84 %（5/271）、100 %（5/5）;桡骨发育不全0.36 %（1/271）、100 %（1/1）;缺肢畸形1.84 %（5/271）、100 %（5/5）;足内翻6.27 %（17/271）、82.35 %（14/17）;手掌畸形1.10 %（3/271）、100 %（3/3）;指趾畸形81.91 %（222/217）、1. 8%（4/222）;多发骨骼畸形6.64 %（18/271）、94.44 %（17/18）。结论：超声对胎儿手掌、脚掌部位以上畸形的检出率较高。指趾畸形出现率最高,但检出率最低。     

Fibroblast growth factors in regenerating limbs of Ambystoma: cloning and semi-quantitative RT-PCR expression studies

Urodele amphibians (newts and salamanders) have the ability to regenerate amputated limbs throughout their life span. Because fibroblast growth factors (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fgf4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanum, and locally collected spotted salamander, Ambystoma maculatum, two salamanders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleotide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showed that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, however, was undetectable by RT-PCR in the distal tips of regenerates, suggesting that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneurogenic and innervated limbs, suggesting that they are not the neurotrophic factors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 upregulation, suggesting Fgf pathways are downstream of nerve-dependence. These data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001.     

山溪鲵的脊柱特征,兼议有尾目脊柱的划分

为探讨有尾目脊椎的划分,本文以小鲵科的山溪鲵(Batrachuperus pinchonii)为例,运用透明骨骼双色法对其脊柱的形态特征进行了观察,并对各部分椎骨特征进行详细描述和绘图.结果显示,山溪鲵的脊椎根据椎骨是否具前关节突、横突、肋骨、肋软骨和脉弓等形态特征可分5部分;同时结合小鲵科其他20种94号标本和蝾螈科6种27号标本的脊柱特征及文献资料,讨论了有尾目脊椎的划分,认为将有尾目脊柱划分为5部分(颈椎、躯椎、荐椎、尾荐椎和尾椎)的观点较将其划分为4部分(颈椎、躯椎、荐椎和尾椎)的观点更合理.     

Ossification patterns in the tetrapod limb – conservation and divergence from morphogenetic events

Two different patterns of the condensation and chondrification of the limbs of tetrapods are known from extensive studies on their early skeletal development. These are on the one hand postaxial dominance in the sequential formation of skeletal elements in amniotes and anurans, and on the other, preaxial dominance in urodeles. The present study investigates the relative sequence of ossification in the fore‐ and hindlimbs of selected tetrapod taxa based on a literature survey in comparison to the patterns of early skeletal development, i.e. mesenchymal condensation and chondrification, representing essential steps in the late stages of tetrapod limb development. This reveals the degree of conservation and divergence of the ossification sequence from early morphogenetic events in the tetrapod limb skeleton. A step‐by‐step recapitulation of condensation and chondrification during the ossification of limbs can clearly be refuted. However, some of the deeper aspects of early skeletal patterning in the limbs, i.e. the general direction of development and sequence of digit formation are conserved, particularly in anamniotes. Amniotes show a weaker coupling of the ossification sequence in the limb skeleton with earlier condensation and chondrification events. The stronger correlation between the sequence of condensation/chondrification and ossification in the limbs of anamniotes may represent a plesiomorphic trait of tetrapods. The pattern of limb ossification across tetrapods also shows that some trends in the sequence of ossification of their limb skeleton are shared by major clades possibly representing phylogenetic signals. This review furthermore concerns the ossification sequence of the limbs of the Palaeozoic temnospondyl amphibian Apateon sp. For the first time this is described in detail and its patterns are compared with those observed in extant taxa. Apateon sp. shares preaxial dominance in limb development with extant salamanders and the specific order of ossification events in the fore‐ and hindlimb of this fossil dissorophoid is almost identical to that of some modern urodeles.     

Genetic parameters and genotype–environment interactions for skeleton deformities and growth traits at different ages on gilthead seabream (Sparus aurata L.) in four Spanish regions

One of the most important problems of fish aquaculture is the high incidence of fish deformities, which are mainly skeletal. In this study, genetic parameters on gilthead seabream (Sparus aurata L.) for skeleton deformities at different ages (179, 269, 389, 539 and 689 days) and their correlations with growth traits were estimated, as were as their genotype × environment interactions (G × E) at harvesting age. A total of 4093 offspring from the mass spawning of three industrial broodstocks belonging to the PROGENSA^® breeding programme were mixed and on‐grown by different production systems in four Spanish regions: Canary Islands (tanks and cage), Andalusia (estuary), Catalonia (cage) and Murcia (cage). Parental assignment was inferred using the standardized SMsa1 microsatellite multiplex PCR. From three broodstocks, 139 breeders contributed to the spawn and a total of 297 full‐sibling families (52 paternal and 53 maternal half‐sibling families) were represented. Heritabilities at different ages were medium for growth traits (0.16–0.48) and vertebral deformities (0.16–0.41), and low for any type of deformity (0.07–0.26), head deformities (0.00–0.05) and lack of operculum (0.06–0.11). The genetic correlations between growth and deformity traits were medium and positive, suggesting that to avoid increasing deformities they should be taken into account in breeding programmes when growth is selected. The G × E interactions among the different facilities were weak for length and deformity and strong for growth rate during this period. These results highlight the potential for the gilthead seabream industry to reduce the prevalence of deformities by genetic improvement tools.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re‐epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration.     

Vitamin A mediated limb deformities in the common Indian toad, Bufo melanostictus (Schneider)

Several types of limb deformities were induced by vitamin A in B. melanostictus. These ranged from total suppression of all the limbs (ectromelia) to partial development of either the forelimb or the hindlimb or both (mesomelia) to reduction or absence of digits in either the forelimbs or hindlimbs or both and absence of long bones in either the forelimbs or hindlimbs or both (phocomelia) or duplication of the hindlimbs (polymelia). All the limb abnormalities were induced in the developing limbs of the tail amputated tadpoles of B. melanostictus following vitamin A treatment, which is all the more interesting. The results suggest that vitamin A induces the above mentioned abnormalities by either switching on or over-expressing or disrupting the limb-specific hox genes by yet unknown mechanisms.     

The spinal cord supports of vertebrae in the crown‐group salamanders (Caudata,Urodela)

The development of spinal cord supports (bony thickenings which extend into the vertebral canal of vertebrae) in primitive (Salamandrella keyserlingii) and derived (Lissotriton vulgaris) salamanders were described. The spinal cord supports develop as the protuberances of periostal bone of the neural arches in the anteroproximal part of the septal collagenous fibers which connect a transverse myoseptum with the notochord and spinal cord, in the septal bundle inside the vertebral canal. Spinal cord supports were also found in some teleostean (Salmo salar, Oncorhynchus mykiss) and dipnoan (Protopterus sp.) fishes. The absence of the spinal cord supports in vertebrates with cartilaginous vertebrae (lampreys, chondrichthyan, and chondrostean fishes) corresponds to the fact that the spinal cord supports are bone structures. The absence of the spinal cord supports in frogs correlates with the lack of the well developed septal bundles inside the vertebral canal. The spinal cord supports are, presumably, a synapomorphic character for salamanders which originated independently of those observed in teleostean and dipnoan fishes. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Evolution of axial patterning in elongate fishes

Within the ray-finned fishes, eel-like (extremely elongate) body forms have evolved multiple times from deeper-bodied forms. Previous studies have shown that elongation of the vertebral column may be associated with an increase in the number of vertebrae, an increase in the length of the vertebral centra, or a combination of both. Because the vertebral column of fishes has at least two anatomically distinct regions (i.e. abdominal and caudal), an increase in the number and relative length of the vertebrae could be region-specific or occur globally across the length of the vertebral column. In the present study, we recorded vertebral counts and measurements of vertebral aspect ratio (vertebral length/width) from museum specimens for 54 species representing seven groups of actinopterygian fishes. We also collected, from published literature, vertebral counts for 813 species from 14 orders of actinopterygian and elasmobranch fishes. We found that the number of vertebrae can increase independently in the abdominal and caudal regions of the vertebral column, but changes in aspect ratio occur similarly in both regions. These findings suggest that abdominal vertebral number, caudal vertebral number, and vertebral aspect ratio are controlled by separate developmental modules. Based on these findings, we suggest some candidate developmental mechanisms that may contribute to vertebral column patterning in fishes. Our study is an example of how comparative anatomical studies of adults can generate testable hypotheses of evolutionary changes in developmental mechanisms.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 97–116.     

Essential mesenchymal role of small GTPase Rac1 in interdigital programmed cell death during limb development

Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.     

Effects of Pollutants on Molting and Regeneration in Crustacea

Many environmental pollutants have toxic effects that can alternormal limb regeneration and molting in Crustacea. The mostcommon effect of heavy metals is that of retardation of regenerationof limbs accompanied by a delay in ecdysis; in some cases regenerationis affected without altering the molt cycle. Chlorophenols anddithiocarbamates caused inhibition of regeneration without affectingmolting in shrimp. Organic toxicants such as aromatic hydrocarbonsanddioxins also result in a decrease in the growth increment permolt, while DDT was found to accelerate regeneration and molting.A number of toxicants also produce morphological alterationsin the regenerated limbs of crabs. These may be relatively minor,such as reduced number of pigment cells, setae, or tuberclesin the regenerated limbs (mercury and cadmium), or may be moremajor deformities, such as abnormal bending in the limb or claw(tributyltin), or defects inchitin formation in the exoskeleton(diflubenzuron).     

Ribeiroia Infection Is Not Responsible for Vermont Amphibian Deformities

Reports of limb deformities in amphibians have garnered wide notice from scientists and the public alike. Recent laboratory and field research has supported the hypothesis that infection by the helminth parasite, Ribeiroia ondatrae, is associated with deformities, particularly in the western United States. In this study, observational and experimental evidence from eastern United States (Vermont) provides evidence that Ribeiroia is absent from a large sample of sites including those with a history of relatively high frequencies of deformity, that the composition of deformities is distinct from that associated with experimental infection by Ribeiroia, and that the composition of limb deformities seen in natural populations in Vermont is typical of that reported in the literature. We suggest that while Ribeiroia has been shown to be responsible for deformities in some species and locations, other factors may be responsible where the composition of deformities is inconsistent with patterns resulting from known Ribeiroia infection.