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Light and electron microscopy shows the osteoderms of Anguis fragilis to be small, flat disks located in the dermis along the adult trunk: microradiography established the extent of the mineralization. Each osteoderm coincides exactly with an epidermal fold forming the keratinized scales characteristic of the skin of reptiles. Sections perpendicular to the surface show two mineralized layers differing in histological and histochemical characteristics and in fine structure, although both contain collagen fibrils. The structure of each layer can be related to that of the surrounding dermis. The outer superficial layer located in the loose dermis contains few collagen bundles that form a discontinuous sheet at the upper surface of the osteoderms. This superficial layer appears to be constituted of units separated by furrows and is composed of woven fibered bone. The basal plate comprises stratified lamellae formed of parallel-oriented collagen fibrils; the fibrils of successive lamellae lie at right angles. The densely packed collagen fibrils of the basal plate are distributed similarly to those of the dense dermis within which it lies. This layer exhibits structural and histochemical characteristics of a lamellar bone. The presence of two different layers in the osteoderms of Anguis fragilis may reflect their mode of formation, which consists of the deposit of mineral crystals in the preexisting dermal tissue. This mineralization process, considered as a “metaplastic ossification,” may reflect the potentiality retained by the dermis of reptiles to form mineralized structures.

Abstract A debate on the determinism (phylogenetic versus functional) of the diversity of bone histological features has centred the interest of bone comparative biologists. While some authors have noticed the presence of a phylogenetic signal in bone tissue variation, many others have argued that these characters may not include much phylogenetic information, but rather reflect functional factors. Here we quantify both components in a sample of amniotes. We hypothesize that: 1/ the observed variation is partly the outcome of shared ancestry (phylogenetic factor) and 2/ for a given quantity of bone produced, tissues formed at a rapid rate may have a higher fraction of vascular cavities than those produced at a slower rate (functional factor). Variation partitioning analyses show that the phylogeny explains a significant portion of the variation of bone vascularity (85.3%), bone growth rate also explains a significant portion of this variation (68.3%), and there is an important overlap (67.9%). Finally, an optimization through least-squares parsimony of bone growth rates onto the phylogeny shows that the most important evolutionary change may have occurred after the split between crocodiles and birds. This change may be linked to the origin of avian endothermic metabolism because high growth rates involve high protein turnover, which is very energy consuming. We conclude that the debate on the dichotomy between phylogenetic versus functional causation of bone histological diversity is misleading, because we have shown that bone vascularity has, at the same time, a functional significance and a phylogenetic signal.

The presence of bone growth marks reflecting annual rhythms in the cortical bone of non-avian tetrapods is now established as a general phenomenon. In contrast, ornithurines (the theropod group including modern birds and their closest relatives) usually grow rapidly in less than a year, such that no annual rhythms are expressed in bone cortices, except scarce growth marks restricted to the outer cortical layer. So far, cyclical growth in modern birds has been restricted to the EoceneDiatryma, the extant parrotAmazona amazonicaand the extinct New Zealand (NZ) moa (Dinornithidae). Here we show the presence of lines of arrested growth in the long bones of the living NZ kiwi (Apteryxspp., Apterygidae). Kiwis take 5–6 years to reach full adult body size, which indicates a delayed maturity and a slow reproductive cycle. Protracted growth probably evolved convergently in moa and kiwi sometime since the Middle Miocene, owing to the severe climatic cooling in the southwest Pacific and the absence of mammalian predators.

The amniotes generally lay eggs on land and are thereby differentiated from lissamphibians (salamanders, frogs and caecilians) by their developmental pattern. Although a number of 330–300-Myr old fossils are regarded as early tetrapods placed close to amniotes on the basis of anatomical data, we still do not know whether their developmental pattern was more similar to those of lissamphibians or amniotes. Here we report palaeohistological and skeletochronological evidence supporting a salamander-like development in the seymouriamorph Discosauriscus . Its long-bone growth pattern, slow diaphyseal growth rate and delayed sexual maturity (at more than 10 years old) are more comparable with growth features of extant salamanders rather than extant amniotes, even though they are mostly hypothesized to be phylogenetically closer to living amniotes than salamanders.

The biological features observed in every living organism are the outcome of three sets of factors: historical (inherited by homology), functional (biological adaptation), and structural (properties inherent to the materials with which organs are constructed, and the morphogenetic rules by which they grow). Integrating them should bring satisfactory causal explanations of empirical data. However, little progress has been accomplished in practice toward this goal, because a methodologically efficient tool was lacking. Here we use a new statistical method of variation partitioning to analyze bone growth in amniotes. (1) Historical component. The variation of bone growth rates contains a significant phylogenetic signal, suggesting that the observed patterns are partly the outcome of shared ancestry. (2) Functional causation. High growth rates, although energy costly, may be adaptive (i.e., they may increase survival rates) in taxa showing short growth periods (e.g., birds). In ectothermic amniotes, low resting metabolic rates may limit the maximum possible growth rates. (3) Structural constraint. Whereas soft tissues grow through a multiplicative process, growth of mineralized tissues is accretionary (additive, i.e., mineralization fronts occur only at free surfaces). Bone growth of many amniotes partially circumvents this constraint: it is achieved not only at the external surface of the bone shaft, but also within cavities included in the bone cortex as it grows centrifugally. Our approach contributes to the unification of historicism, functionalism, and structuralism toward a more integrated evolutionary biology.

We explored the factors that explain the variation in resting metabolic rates (RMR) in growing amniotes by using the phylogenetic comparative method. For this, we measured raw RMR (mL O2 h−1), body mass, body mass growth rate, and periosteal bone growth rate in a sample of 44 growing individuals belonging to 13 species of amniotes. We performed variation partitioning analyses, which showed that phylogeny explains a significant fraction of the variation of mass-specific RMR (mL O2 h−1 g−1), and that the cost of growth is much higher than the cost of maintenance. Moreover, we tested the hypothesis of the independence of energy allocation, and found that maintenance metabolism and growth rates are not significantly related. Finally, we calculated the statistical parameters of the relationship between geometry-corrected RMR (mL O2 h−1 g−0.67) and bone growth rate. This relationship could potentially be used in palaeobiology to infer RMR from bone tissue samples of fossil species by assuming Amprino’s rule (according to which bone tissue types reflect bone growth rates). These estimates would be especially interesting for Mesozoic non-avian theropod dinosaurs and Permian and Triassic therapsids to investigate, respectively, the origin of avian and mammalian endothermy. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92, 63–76.

A parsimony optimization of the presence of high-frequency flapping flight onto a phylogeny of 29 species of birds shows that this is a derived character state that has been acquired at least four independent times: by the last common ancestor of Alcidae, that of Podicipedidae, that of Anatidae, and that of Rallidae. Cineradiographic analysis has shown that the furculae of birds underwent extraordinary deformations during the wingbeat cycle. Cyclical deformations are known to produce microfractures in the bone tissue, which may be a stimulus for Haversian remodelling, a mechanism of resorption and reconstruction of bone tissue that may repair bone microdamage. In the present study, we performed a comparative analysis in a phylogenetic context to test the effect of the frequency of cyclical deformations and body mass on the rate of Haversian remodelling in the furculae of birds. A variation partitioning analysis showed that the type of flight (high-frequency flapping flight vs. other kinds of flight of lower wing beat frequency) and body mass explained a significant portion of Haversian bone density (the outcome of Haversian remodelling) and that the phylogeny also explained a significant part of this variation. This phylogenetic signal on Haversian bone density variation may be the outcome of phylogenetic signal on the proximate causes producing Haversian remodelling. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 729–738.

We evaluated ages of larvae in southern Appalachian populations of the salamanders Gyrinophilus porphyriticus and Pseudotriton ruber by the method of skeletochronology applied to femurs. In P. ruber, the method yielded a strong correlation between snout-vent length and age and provided estimates in agreement with earlier studies of larval growth and age that were based on size-frequency data. However, in G. porphyriticus, the correlation between snout-vent length and age was weak and nonsignificant. This result was in accordance with previous studies that showed that larvae of this species do not sort by size into discrete age classes. The failure of skeletochronology in G. porphyriticus was attributed to either extreme variation in growth rate or to some deficiency of the methodology itself when applied to this species. We suggest that larvae of G. porphyriticus may not develop well-defined annual bone layers because of their adaptation to relatively aseasonal, subterranean microhabitats in headwater springs. In contrast, larvae of P. ruber, although overlapping in macrohabitat with G. porphyriticus, are essentially surface-dwellers that are exposed to the seasonal climatic fluctuations of the region.

In spite of the fact that the potential usefulness of bone histology in systematics has been discussed for over one and a half centuries, the presence of a phylogenetic signal in the variation of histological characters has rarely been assessed. A quantitative assessment of phylogenetic signal in bone histological characters could provide a justification for performing optimizations of these traits onto independently generated phylogenetic trees (as has been done in recent years). Here we present an investigation on the quantification of the phylogenetic signal in the following bone histological, microanatomical, and morphological traits in a sample of femora of 35 species of sauropsids: vascular density, vascular orientation, index of Haversian remodeling, cortical thickness, and cross-sectional area (bone size). For this purpose, we use two methods, regressions on distance matrices tested for significance using permutations (a Mantel test) and random tree length distribution. Within sauropsids, these bone microstructural traits have an optimal systematic value in archosaurs. In this taxon, a Mantel test shows that the phylogeny explains 81.8% of the variation of bone size and 86.2% of the variation of cortical thickness. In contrast, a Mantel test suggests that the phylogenetic signal in histological traits is weak: although the phylogeny explains 18.7% of the variation of vascular density in archosaurs, the phylogenetic signal is not significant either for vascular orientation or for the index of Haversian remodeling. However, Mantel tests seem to underestimate the proportion of variance of the dependent character explained by the phylogeny, as suggested by a PVR (phylogenetic eigenvector) analysis. We also deal with some complementary questions. First, we evaluate the functional dependence of bone vascular density on bone size by using phylogenetically independent contrasts. Second, we perform a variation partitioning analysis and show that the phylogenetic signal in bone vascular density is not a by-product of phylogentic signal in bone size. Finally, we analyze the evolution of cortical thickness in diapsids by using an optimization by squared change parsimony and discuss the functional significance of this character in terms of decreased buoyancy in crocodiles and mass saving in birds. These results are placed in the framework of the constructional morphology model, according to which the variation of a character in a clade has a historical (phylogenetic) component, a functional (adaptive) component, and a structural (architectural) component.

We studied the age structure and the growth of a breeding population of parsley frogs, Pelodytes punctatus, from a pond system in northern Spain using skeletochronological methods. This population lives in an upland area with marked seasonal differences characterized by long, cold winters and dry summers. We assessed, using the mark–recapture method, the annual periodicity of bone growth marks. Only one line of arrested growth is laid down per winter, showing, in general, a well-defined histological structure. Females are larger than males with 43.31 and 36.32 mm average body length, respectively. This sexual dimorphism in body length increases with age. Males and females reach sexual maturity when they are 1 year old. The oldest males were 8 years old while the females were 2 years older. Growth curves for both sexes were constructed based on body size and the number of lines of arrested growth found in bone sections. The growth rate of males declined abruptly after the first year. Females delayed reprod...

Some aspects of the historical development of paleohistology in vertebrate paleontology are briefly reviewed, with emphasis on studies of fossil amphibian and reptile bone tissues, as compared with thoses of extant forms. A consideration of bone tissue diversity and of its likely determinism lead to its value as a direct tool in systematics beeing questioned in both extant and extinct forms. Rather, it is suggested that its variations may be used as a biological recorder of ontogenies sensu lato, offering data on species or populations‐specific life history traits and even hints at their larger scale evolution among extinct lineages.

During the summer of 1997, 115 Plethodon metcalfi were collected on a 10-year-old clearcut (50) and in a nearby forest (65) in the vicinity of Highlands, North Carolina. Sex, reproductive status, snout–vent length, mass, and age were used to assess the effects of clearcutting. Relative to the forest population, the clearcut population had a smaller proportion of juveniles and proportionately fewer adult males in reproductive condition. Mature female salamanders had greater snout–vent lengths (SVL) than mature males, but there was no difference in average SVL between sites. Masses of mature salamanders did not differ by sex, but salamanders on the clearcut were more massive than their counterparts in forest. Ages of mature salamanders did not differ by sex or site. These results suggest that age distributions, masses, and reproductive efforts of P. metcalfi populations on clearcuts in the southern Blue Ridge Mountains are altered by clearcutting. Future research concerning effects of forest manage...

We evaluated age structure in three species of desmognathine salamanders at Coweeta Hydrologic Laboratory, North Carolina, for comparison with the results of an earlier study of age structure in these species at a nearby locality (Wolf Creek, North Carolina). At Coweeta, both Desmognathus quadramaculatus and D. monticola attain larger adult body sizes and greater ages than their Wolf Creek counterparts. The differences appear to stem in part from greater ages at sexual maturation in both species at Coweeta versus Wolf Creek. This disparity is most pronounced in D. quadramaculatus, where the estimated ages at first reproduction are 7–8 yr in males and 9–10 yr in females. The Coweeta populations of D. quadramaculatus and D. monticola may also have higher survivorship and higher rates of post-maturational growth than those at Wolf Creek. In contrast, in the third and smallest species, D. ocoee, the Coweeta population did not differ in size and age attributes from that at Wolf Creek. We hypothesize that the m...

International audience; We describe the age structures of two neighbouring terrestrial salamander populations. The skeletochronological method was also used on larvae in utero and on new-born individuals. The age of adults was 8-24 years in population A, while males reached maturity at 3-5 years old and the youngest females were 6 years old in population B. Males and females from population B were also larger than those in population A. For the first time, lines of arrested growth (LAGs) were also found in the humerus of intra-uterine larvae and new-born individuals, indicating that young can spend up to 3 years in utero (population B) and up to 4 years (population A) before hatching. Growth of adults (fitted by the Bertalanffy model) also exhibited differences in growth coefficient (k) and mean asymptotic length (SVLmax) between sexes and populations. Local climatic conditions differed between the two areas of these populations and we hypothesize that the number of rainy days directly influences foraging during the short period of activity (< 3 months), leading to a delay in age at maturity, smaller length and growth rate, and increased gestation duration in the drier environment. The discussion is focused on proximate environmental influences on the variation of length and associated life-history traits in ectotherms, especially in terrestrial salamanders.

Skeletochronology, used to assess some life history traits in the viviparous African toad, enables us to determine that longevity in natura does not exceed 5 years in females and reaches at least 2 years in males. After sexual maturity, reached at four months, the rate of osteogenesis decreases, as probably does body growth. In this study we discovered a strong endosteal bone resorption in long bone cortices in both males and females. This phenomena only occurs during the first resting period. This period occurs from October (individuals of 5 months old) to March (individuals of 10 months old). During this time toads are underground and their metabolism is thought to be low. The origin of such an endosteal resorption remains more or less unknown.

Genital maturation of male monitor lizards, a subject poorly documented up to now in spite of the heavy exploitation of these animals, was studied on a sample of 211 Nile monitors (Varanus niloticus) from Chad (Lake Chad), Mali (central delta of the Niger), and Nigeria (Hadejia-N'Guru wetlands). Snout-vent lengths (LC) in the sample ranged from 15.6 to 85.5 cm. After a morphometric study of the monitors, their testes were sampled, weighed at ±0.01 g and fixed for histology. The age of each specimen was estimated by skeletochronology. The testes of monitors less than 36 cm LC (Malian specimens) or 40 cm LC (Chadian and Nigerian individuals) do not contain mature spermatozoa, whereas the testes of larger specimens do. Most Malian monitors aged 18 months (1.5 years) have mature spermatozoa. Conversely, in Chadian and Nigerian individuals, mature gametes appear only in specimens aged 30 months (2.5 years). These data indicate that genital maturity in the Nile monitor is reached at 36-40 cm LC and 1.5-2.5 years according to the populations. The growth in mass of the testes, compared with LC, is an accurate indicator of body size at sexual maturity, and confirms the precocity of Malian specimens. Conversely, testis growth is poorly related to age. These results are discussed with reference to the possible accommodation of Nile monitor populations to heavy exploitation by means of precocious recruitment of the male breeding stock.

Large amounts of vertebrate remains regurgitated by owls are potentially available for continuous monitoring of predated populations, without human interference or the need to sacrifice any specimens. Presumably, Pleistocene and Holocene fossil microvertebrate assemblages found in caves or shelters are also principally the result of predation by nocturnal birds. Our study tests, for the first time, the standard skeletochronological technique on such prey remains. We analyse two Spanish samples from the genus Discoglossus (Amphibia, Anura, Discoglossidae), a living population of D. galganoi (n = 65 individuals) predated by the European Barn Owl, Tyto alba, and a Pliocene-Pleistocene sample of an extinct species (n = 22) presumably predated by Tyto balearica, a closely related ancestor of T. alba. The histological peculiarities and alterations found in bones of both samples are described, but the results indicate that, in general, this type of material is suitable for skeletochronological applications. In the living population, both size and longevity were greater than previously recorded for the species. For both samples, size and age are positively, though weakly, correlated. Comparisons by age-class show that the extinct species is larger than the living one, particularly for 4- and 5-year-old individuals. Growth rates in juveniles (or larvae) of the extinct species should have been slightly higher than in the living species, with similar growth rates after the third year. Recorded longevity is higher in the extinct species. All these life-history traits support a distinct taxonomic status for the extinct Balearic Discoglossus species.

The structure and the histological expression of annual bone growth marks of Iberian water frogs (Rana perezi) from a warm temperate climate region (southwestern Spain) differ markedly from the pattern observed in colder climate populations. Forty-eight percent of winter growth marks appeared to be annuli; 52% are lines of arrested growth but 20% of these are weakly expressed. Annuli or lines of arrested growth were very diffuse or absent in 7% of the 103 individuals studied, suggesting that variations in climate have a stronger influence on growth-mark formation than was previously considered. Males and females are sexually mature in their second year, although some males mature at 1 year of age and some females at 3 years. The oldest males were 4 years old, 1 year younger than males from northern populations. The oldest female was 6 years old. The wide range of body sizes among the 1-year-old froglets can be explained by the extended period of metamorphosis, from July to November.

To specify the influence of a permanent aquatic life on skeletal mass and bone structure in anuran amphibians, the general and regional masses of the skeleton, as well as several histomorphometric indices of the bones (porosity, corticodiaphyseal index, density) were compared in four ranid species, two predominantly aquatic (Rana perezi and R. esculenta) and two more terrestrial (R. temporaria and R. dalmatina). The relative mass of the skeleton (as a percentage of the total body mass) is significantly lower in aquatic forms. This difference is not a phenomenon of intra- or inter-specific allometry between skeletal mass and body mass (size effect). Rather, it reflects a selective reduction of skeletal volume (low corticodiaphyseal index values and higher porosity), in particular in the autopodia, pectoral girdle, front limbs, and vertebral column of aquatic forms. These results reinforce the idea that habitat and mode of life are selective factors producing a more or less developed skeleton in anurans. Whether a lighter skeleton is a permanent adaptative feature or a reversible response in predominantly aquatic anurans remains to be tested.

Abstract Water fluxes and daily energy expenditure (DEE) of Gallotia galloti, G. stehlini and G. atlantica, were estimated over a three-year period using the doubly-labelled water (DLW) method. Water influx varied little between seasons and between sexual categories. Juveniles tended to have higher water fluxes in spring in all three species; after a dry period the water turnover tended to decrease for all sexes in G. galloti and G. stehlini, whereas little variation was observed for G. atlantica. The average water influx, combined for all periods, was 46.27, 50.97 and 38.20 ml H2O.kg-1 d-1 for the three species respectively; only the last value differs significantly from the remaining two. The mean DEE, for all periods combined, were 189.7, 179.4 and 146.5 J g-1 d-1 for the three species respectively. As for water turnover, only the value for G. atlantica differed significantly. These data suggest that: G. atlantica may be better adaptated to maintain homeostasis during dry periods and that differences in interspecific DEE can also be explained by others factors than differences in habitat, climatic conditions, daily profiles of activity or body temperatures. We suspect that the incidence of intraspecific competition has more importance in G. atlantica than in the two other species.

Changes in age structure of local populations were analyzed to determine the migrations of adult crested newts (Triturus cristatus) in a network of ponds. An individual's age was estimated by applying a skeletochronological method to the phalanges. The local populations studied bred in four neighbouring cattle ponds, the distance between any two of which did not exceed 150 m. These ponds belong to the larger network. Though the size of the whole population doubled from one year to the next, one local population became extinct due to lack of recruitment of young breeders. The larger local population attracted not only young breeders but also older newts, which probably came from other ponds. In ponds inhabited by fish, the newts were mainly young animals (2–3 years old), suggesting that they leave such ponds after their first breeding season. The results are discussed with regard to the model proposed by Gill for metapopulations of Notophthalmus viridescens. In the crested newt, contrary to Gill's predictions, all the sites do not recruit young breeders, and fidelity to the breeding site may be broken. The two species differ in longevity and age at maturity, hence it is suggested that functional models of metapopulations must integrate life-history parameters.

Abstract We conducted a comparative study of some biological parameters between different allopatric northern (high and low altitudes) and southern (low altitude) populations of Triturus marmoratus in Portugal. Individual age was determined by counting lines of arrested growth in histological sections of the humerus and phalanx. This study documents interpopulational differences in longevity, age of sexual maturity and growth of individuals. Individuals from high altitudes in the north reached older ages than those from low altitudes but both reached older ages than those from the south. Maturity was reached sooner among the southern newts for both sexes when compared to the northern populations. The southern newts also reached their sexual maturity at smaller mean sizes than the northern ones. We interpret these differences and suggest how genetical and epigenetical components can be both involved and account for the observed variability.

L'immersion des civelles (Anguilla anguilla L.) dans une solution hyperosmotique (5 % de chlorure de sodium) pendant 3'30 puis dans une solution contenant 1 % de chlorhydrate de tetracycline (CHTC) ou 2 % de fluorescéine (DCAF) pendant encore 3'30, entraîne une accélération de la vitesse d'entrée de ces deux marqueurs vitaux dans l'organisme des poissons et un marquage rapide des organes calcifiés (vertèbres et otolithes). Des résultats similaires sont obtenus en plaçant les civelles dans une solution contenant à la fois 5 % de chlorure de sodium et 1 % de CHTC ou 2 % de DCAF pendant 3'30. Le choc osmotique est proposé comme une avancée technique pour le marquage vital en masse des larves, des alevins et des stades juvéniles car il permet d'abaisser considérablement le temps de contact nécessaire entre les poissons et la solution de fluoromarqueur. Nous présentons une application de cette technique pour le marquage de 500 kg de civelles avec de la tetracycline. La réalisation de cette manipulation a demandé une journée et demie de travail à 5 personnes. Les résultats obtenus sont de très bonne qualité puisque l'on obtient 100 % de spécimens marqués dont 70 % le sont fortement. Il est donc possible de marquer une grande quantité de civelles dans un laps de temps réduit. Le problème de la reconnaissance in situ des animaux ainsi marqués est discuté.

The biological features observed in every living organism are the outcome of three sets of factors: historical (inherited by homology), functional (biological adaptation), and structural (properties inherent to the materials with which organs are constructed, and the morphogenetic rules by which they grow). Integrating them should bring satisfactory causal explanations of empirical data. However, little progress has been accomplished in practice toward this goal, because a methodologically efficient tool was lacking. Here we use a new statistical method of variation partitioning to analyze bone growth in amniotes. (1) Historical component. The variation of bone growth rates contains a significant phylogenetic signal, suggesting that the observed patterns are partly the outcome of shared ancestry. (2) Functional causation. High growth rates, although energy costly, may be adaptive (i.e., they may increase survival rates) in taxa showing short growth periods (e.g., birds). In ectothermic amniotes, low resting metabolic rates may limit the maximum possible growth rates. (3) Structural constraint. Whereas soft tissues grow through a multiplicative process, growth of mineralized tissues is accretionary (additive, i.e., mineralization fronts occur only at free surfaces). Bone growth of many amniotes partially circumvents this constraint: it is achieved not only at the external surface of the bone shaft, but also within cavities included in the bone cortex as it grows centrifugally. Our approach contributes to the unification of historicism, functionalism, and structuralism toward a more integrated evolutionary biology.

New material from a small, presumably adult enantiornithine bird (Saurischia: Aves) from the Lower Cretaceous of Las Hoyas (Spain) provides new data on the bone histology of those Mesozoic birds, which became extinct at the Cretaceous–Tertiary boundary. The material is referred to cf. Concornis lacustris. The outer cortex of the studied long bones is formed of a rather typical parallel-fibered bone tissue, with sparse flattened osteocyte lacunae, evidence of lines of arrested growth (LAGs) and no vascularization. This structure matches the outer circumferential layer (OCL) of many extant mature, small to tiny birds and also agrees with earlier descriptions of enantiornithine cortical bone. The deeper cortex progressively contains a much more plentiful component of osteocyte lacunae, which are more plump than flat. This region is also permeated by a few vascular canals surrounded by bone lamellae that form osteonal structures. The relationship of the osteonal material to the neighbouring primary tissue strongly suggests that the osteons are secondary. In some sections, it is possible to follow those osteons as formed by outward extensions from the endosteal bone tissue into the deep primary cortex. This specimen shows more histodiversity than the few enantiornithine samples previously reported. This further suggests that although growth may have been slow when the adult size was approached, it was more rapid at earlier stages. Even if as yet not enough is known of those early stages to document just how rapid early growth may have been, and whether enantiornithines grew differently from the basal avian models known from other early birds, the new data do not necessarily suggest that they had growth regimes quite distinct from extant birds of similar body sizes nor, by implication, completely different metabolic patterns or regimes. To cite this article: O. Cambra-Moo et al., C. R. Palevol 5 (2006).

Because they are mineralised, skeletal tissues can record and preserve indefinitely in their microstructures the expression of various constraints and especially the passage of time. Such traces of time can be either continuous or periodic (growth marks), but when deciphered they offer a powerful tool to reconstruct life history traits and even ecological conditions of time in extant as well as extinct species. Nevertheless, the temporal message ‘printed’ in skeletal tissues can be disturbed or even destroyed by various causes that need to be understood before this ‘biological chronometer’ can be accurately used. To cite this article: J. Castanet, C. R. Palevol 5 (2006).

The histological features of mineralized tissues can be preserved for hundreds of millions of years, and are therefore important potential sources of information for reconstructing the life history traits of extinct species. Bone growth rates and the duration of the growth period have recently been estimated in fossil archosaurs from periosteal ossification (a mechanism responsible for bone diametral growth). Similarly, data on endochondral ossification (the mechanism responsible for bone longitudinal growth) may also yield information on growth duration and rate among extinct vertebrates, as long as potentially informative structures are preserved. However, in order to carry out palaeobiological estimations of growth rate and/or the duration of growth, it is first necessary to quantify in extant species the relationship between these life history traits and the histological features of endochondral ossification that are potentially preserved in the fossil record. Here we analyse the ontogenetic variation of both bone longitudinal growth rate and the thickness of the calcified cartilage in the femora of two Galloanserae (Aves) and find a significant positive relationship between these variables in both species. We discuss possible factors underlying interspecific differences in this relationship, and conclude that it could be applied with caution to draw palaeobiological inferences.

Here we study the occurrence of torsion-resisting morphological and histological features (thin bone walls, circular shaft cross-section, oblique collagen fibers, and laminar tissue arrangement) in a sample of 168 long bones from wings and legs of 22 bird species. These structural parameters were measured in mid diaphyseal undemineralized cross-sections and analyzed using uni-, bi-, and multivariate (principal components analysis) data analysis techniques. We found that the four variables are significantly and positively correlated, and that covariation between variables accounts for as much as 58% of the total variation. These results suggest that torsion is a main determinant of the macro- and microstructural design of long bones in birds. Humerus, ulna, and femur generally possess torsion-resisting features, while other bones (radius, carpometacarpus, tibiotarsus, tarsometatarsus, and foot phalanx) rather show bending/axial load-resisting structural properties. These results are congruent with in vivo strain data from the literature, which reported high torsional loading in humerus and ulna during flapping flight, but also in the subhorizontal avian femur during terrestrial locomotion. The precise function of the laminar tissue spatial arrangement, the role of pneumatization, and the influence of flight mode are discussed.

SUMMARY Microstructure–function relationships remain poorly understood in primary bone tissues. The relationship between bone growth rate and bone tissue type, although documented in some species by previous works, remains somewhat unclear and controversial. We assessed this relationship in a species with extreme adaptations, the king penguin (Aptenodytes patagonicus). These birds have a peculiar growth, interrupted 3 months after hatching by the austral winter. Before this interruption, chicks undergo extremely rapid statural and ponderal growth. We recorded experimentally (by means of fluorescent labelling) the growth rate of bone tissue in four long bones(humerus, radius, femur and tibiotarsus) of four king penguin chicks during their fastest phase of growth (3–5 weeks after hatching) and identified the associated bone tissue types (`laminar', `longitudinal', `reticular' or`radial' fibro-lamellar bone tissue). We found the highest bone tissue growth rate known to date, up to 171 μm day–1 (mean 55 μm day–1). There was a highly significant relationship between bone tissue type and growth rate (P

In primates, age determination using lines of arrested growth (LAGs) from bones has rarely been attempted, and the reliability of these structures has never been experimentally validated. In order to test skeletochronology in primates, LAGs were studied mainly in the long bones of known age Microcebus murinus, a small primate, whose potential longevity may reach 12 years. LAGs were extensively studied in 43 males and 23 females ranging from juveniles to 11-year-old adults. All individuals were born and reared in captivity. Some young individuals were injected with fluorescent dyes to quantify bone growth rates. LAGs in the diaphysis of the tibia are well correlated with age and this skeletal element appears to be the best for assessing skeletochronology in Microcebus murinus. There is strong evidence that the seasonal cycle of photoperiodicity is more important than age alone in producing LAGs.

The metoposaurid Dutuitosaurus ouazzoui, from the Carnian (Upper Triassic) of southern Morocco is one of the best known temnospondyls; more than 75 specimens have been found, including 15 sub-complete skeletons of various sizes from ImiN'Tanoute, a mass mortality locality (Bull. Soc. Geol. Fr. 25 (1983) 623) in the Argana Basin, Western High Atlas. Ten femora, sampled from sub-complete skeletons belonging to a growth series, have been sectioned at the mid-diaphyseal level in order to perform histological and skeletochronological analyses. Sections from juveniles and adults show microstructures typical of aquatic stegocephalians (absence of a free medullary cavity, presence of an extensive spongiosa that merges gradually into a cortical compacta). Bone sections from juveniles reveal a relatively high initial growth rate (no secondary osteons, cortex with wide and densely vascularized growth zones, alternating with thin annuli or Lines of Arrested Growth—LAGs). Bone sections from adults indicate a decreased growth rate (less vascularized external cortex, osteocyte lacunae parallel to the cortical stratification), probably after the acquisition of sexual maturity, and a moderate amount of remodeling (presence of a few secondary osteons, of erosion bays in the cortical compacta). Growth marks are correlated with environmental changes, likely to be seasonal cycles. Compactness profiles have been quantified using ‘Bone profiler’. Comparison with profiles of extant taxa confirms the hypothesis that Dutuitosaurus was aquatic.

Periosteal bone histology expresses its rate of deposition. This fundamental relationship between bone structure and growth dynamics, first assumed by Amprino many decades ago, was quantified in preliminary studies, but never statistically tested. Moreover, the precise typological characters of bone tissue linked to growth rate remained poorly known. Here, we present the first statistical analysis of 'Amprino's rule', measured on comprehensive growth series of the mallard, Anas platyrhynchos. Growth rates were assessed by fluorescent labelling. Bone typology was described according to Ricqlès' typological classification. Results show that the presence and proportion of primary osteons, two consequences of bone initial porosity at the time of its deposit, are strongly related to bone growth rate. However, no significant relationship between primary osteons orientation and bone growth rate could be detected, at least for osteonal orientations (longitudinal, laminar and reticular) and growth rates values observed in mallard long bones. These results suggest that Amprino's rule holds for some major typological characters of primary compact bone tissues (i.e. primary osteons presence and proportion). However, it is irrelevant to some other characters (i.e. osteonal orientation), the meaning of which remains to be discovered.

The first quantitative experimental data on growth dynamics of the primary cortical bone of young ratites demonstrate the following. 1) From hatching to 2 months of age, cortical thickness remains constant, thereby expressing equilibrium between periosteal bone deposition and an endosteal bone resorption. 2) Radial growth rates of the diaphyseal bone cortex are high (10–40 μm·day –1 on average – maximum 80 μm·day –1 ) in the hindlimb (femur, tibiotarsus and tarsometatarsus). Wing bones are smaller and later developed. They have lower rates of radial osteogenesis (2–14 μm·day –1 ). 3) High growth rates are linked to densely vascularized primary bone belonging to the reticular or laminar tissue types. Growth rates fall when bone vascular density decreases. These results emphasize the importance of examining a large number of skeletal elements in order to build a precise knowledge of the general relationship between bone growth rate and bone tissue type. They also stress the potential of bone growth rate quantification among extinct tetrapods, including non-avian dinosaurs.

The value of skeletal cyclic growth marks, or SGMs, for age estimation in the Nile monitor, Varanus niloticus, a species highly exploited for food and leather, was checked by a mark-recapture experi- ment including in vivo labelling of bone growth. The basic histological features of the SGMs are described, as well as the variability in the structure of these marks with regard to the skeletal elements considered, the technique for processing the sections, and differences between populations or individuals. Ground sec- tions from the shaft of the fibula, observed under transmitted polarized light, are best suited for counting growth marks. Bone labelling indicates that SGMs have an annual periodicity and can thus be reliable age indicators as long as somatic growth proceeds. The formation of SGMs is interpreted with reference to the ecology of the Nile monitors. The practical value of skeletochronology for population surveys is discussed. The Nile monitor, Varanus niloticus, is among the most exploited species of tetrapods in Af- rica, especially in the part of its range corre- sponding to the sudano-tropical zone (part of the continent between 10 and 16?N) where it is used as food and for the leather trade. Ten years ago, the number of skins of this species legally exported by just four countries was close to one million specimens per year (Luxmoore and Groombridge, 1990; de Buffrenil, 1993). Though

We studied sexual size dimorphism is in two breeding populations of midwife toads (Alytes obstetricans and Alytes cisterasii) from which skeletochronological data provide information on individual age. The population values show that females are larger than males in both species (A. obstet- ricans, SVLmale = 41.74 mm, SVLfemale = 49.69 mm; A. cisternasii, SVLmale = 35.79, SVLfeale = 38.58). The values obtained for population size dimorphism (Ln(xmale)-Ln(xfemale) -0.174 for A. obstetricans and -0.074 for A. cisternasii) are are higher than the age-specific values (range -0.126 to -0.055 for A. obstet- ricans; -0.101 to -0.043 for A. cisterasii). Differences between age-specific and population dimorphism values are particularly marked in A. obstetricans, where the age distribution of the sample of males and females differ the most. This result suggests that caution should be used when considering population data on size dimorphism to test evolutionary hypotheses about the evolution of this phenomenon. The dimorphism values obtained for the two species are not markedly different from those obtained from other temperate anurans without male parental care. This result does not support the prediction of the invest- ment hypothesis (Williams, 1966; Trivers, 1972) that male parental care may act as a limitation on sexual selection on male size.

The age of specimens of the plethodontid salamander Batrachoseps attenuatus was estimated using skeletochronology. This species differs from other urodeles in that it develops directly within the egg capsule, with no larval period, and it is active in the winter rather than in the summer. Although lines of arrested growth are somewhat irregular and more difficult to interpret than in other species of salamanders studied, age of most individuals was estimated with a high degree of confidence. Maximum adult size in the population studied was about 50 mm snout-vent length. Specimens larger than 40 mm in length ranged in age from four to eight years. Once sexual maturity is achieved, size is a poor estimator of age in this species. In amphibians and reptiles in which annual growth is cyclical, with alternating periods of relatively rapid and relatively slow growth, skeletochronology can be used to estimate the age of individual organisms (Castanet et al., 1993). Cyclical growth results in the production of growth marks in bones and some other hard tissues, and information provided by the annular pattern can be used to obtain accurate estimates of age in amphibians and reptiles (e.g., 60 This content downloaded from 207.46.13.105 on Wed, 25 May 2016 06:43:25 UTC All use subject to http://about.jstor.org/terms SKELETOCHRONOLOGY OF THE SALAMANDER BATRACHOSEPS Castanet and Smirina, 1990). Periodicity in growth is revealed in the histology of the bone, with broad, less dense zones being associated with periods of rapid growth, and narrower, more dense zones called annuli (sensu Peabody, 1961) being associated with periods of decreased growth rates. These latter periods are manifest in distinctive layers of increased density known as lines of arrested growth (LAG). One broad layer plus one LAG generally constitute a complete cycle of growth, typically annual for amphibians that live in regions with predictable climatic cycles of warm and cold, or wet and dry seasons. However, under certain climatic conditions two LAG may be produced in a single year (Caetano et al., 1985). Studies of marked individual newts (Triturus cristatus) of known initial age that extended over several years demonstrated that each LAG corresponds to a winter period of inactivity, and that each broad zone corresponds to the preceding period of summer activity (e.g., Francillon, 1979, 1980; Caetano and Castanet, 1987). Because of these experimental demonstrations of the utility of the method, skeletochronology has become widely used by ecologists studying salamander populations (e.g., Smirina and Sofianidu, 1985; Verrell and Francillon, 1986; Houck and Francillon-Vieillot, 1988; Guyetant et al., 1992; Francillon-Vieillot et al., 1990; Montori, 1990; Flageole and Leclair, 1992; Miaud et al., 1993). When conducting comparative studies of the relation of phylogeny and ontogeny, the relation between size and age is of special importance (Alberch et al., 1979). When age is not determined, size is sometimes substituted, because during the main growth phase there is a general correlation between size and age. However, following attainment of sexual maturity the correlation of size and age is suspect, and may be entirely absent. The present study was undertaken as a component of comparative studies of the ontogeny of salamanders that lack a larval stage and undergo encapsulated development, hatching from the egg as miniature adults (there is no metamorphosis). Prior skeletochronological studies have been biased toward members of the families Salamandridae and Ambystomatidae. The species studied have a biphasic life cycle with a larval period usually of one year or less and a discrete metamorphosis. We studied a population of the California slender salamander, Batrachoseps attenuatus, a miniaturized species that develops directly within the egg capsule. This species has an activity pattern very different than that of European newts and American ambystomatids that have been the subject of skeletochronological studies published to date. Batrachoseps is active only during the winter rainy period, a time of relatively low temperature, and is inactive during the hot, dry summer. It is important to learn if standard skeletochronological techniques can be applied in such situations, because over 50% of the species of salamanders are direct developing and most of these occur in tropical environments which have patterns of seasonality (typically wet-dry) that differ from the north temperate (cold-warm) environments of salamanders studied to date. MATERIALS AND METHODS Salamanders were collected from the field in Berkeley, Alameda Co., California, in January, 1988, and were kept in the laboratory at seasonal light conditions and 14 C from two to eleven months as a component of a study of tooth replacement. When sacrificed, specimens were fixed in 10% buffered formalin and then transferred to 70% ethanol for storage. The specimens studied were measured (from snout to posterior end of vent, SVL) and sexed. In addition to 23 adults, two small specimens ca. 12 mm SVL and in their first year of life were examined. Specimens were prepared for skeletochronological study following standard techniques (Caetano et al., 1985). Frozen sections of demineralized bones (femur and humerus) were cut at 15 micrometers and stained in Ehrlich's hematoxylin. Sections from the midshaft of the diaphysis were mounted in an aquamounting medium, examined by normal micrography and photographed at a constant magnification for analysis of age and growth. The perimeter of each LAG was measured with a curvimeter. Initial counts of LAG were done separately ("blindly") by each author, and results were then compared and final ages assigned. There was high general agreement, but a few instances required consultation and reevaluation. Finally, both authors agreed on all assignments.

Spatial organization of collagenous fibrils in the basal plate of elasmoid scales has been studied in 36 species among 24 teleost families which include the whole order. Angle measurements of the various fibrillary directions were performed on fracture surfaces in frozen scales observed with the scanning electron microscope. The organization of the basal plate is arrayed in three basic patterns. The most remarkable one, found in the more “primitive” families, is a double twisted plywood with a left-handed rotation of the fibrillary directions. The remaining two are an orthogonal plywood, more or less regular, and an “intermediate” plywood, both found among the more “advanced” families. However, an orthogonal plywood is also observed in some “primitive” families, for example in Clupeidae and Salmonidae. We tentatively interpret the left-handed double twisted plywood of the lower teleosteans and of Amiidae as a synapomorphy in relation to the primitive condition of an actinopterygian lineage. On the other hand, within the teleosts, we would consider that the double twisted plywood is a plesiomorphic condition and that the orthogonal plywood is an apomorphy among the “advanced” teleosts families.

After a preliminary descriptive study of the “skeletal growth marks” often used among snakes as an empirical age index, this paper deals with the first experimental results. (1) On Vipera aspis grown from birth under controlled, constant conditions and hence of known age, the number of skeletal growth marks is closely related to animal size, but this size has no relationship with individual age. (2) On Vipera aspis submitted to seasonal variations under experimental conditions and bone labelled with fluorochromes, the number of growth marks added to the skeleton during the experimental study (two years) seems to be in accordance with this duration. This would demonstrate, at least theoretically, a relationship between skeletal growth marks and individual age, as well as incidences of environment conditions. These results are discussed. Resume Apres une etude preliminaire descriptive sur la structure des marques squelettiques de croissance qui, chez les serpents sont utilisees de facon empirique comme indicateur de l'âge, le present travail fait etat de premiers resultats experimentaux. (1) Chez des Viperes aspic elevees depuis leur naissance dans des conditions constantes, controlees (il s'agit donc d'animaux d'âge connu), le nombre de marques squelettiques est etroitement liea la taille des animaux, mais comme elle, n'a plus dans ces conditions experimentales de rapport avec l'âge. (2) Chez des Viperes subissant leur cycle saisonnier sous conditions experimentales et dont on marque in vivo par fluorochromes le tissu osseux, le nombre de marques squelettiques mis en place pendant la duree de l'experience (2 ans) correspond sensiblement a cette duree. Ceci tend a confirmer, en theorie, la relation entre les marques squelettiques et l'âge individuel et son interaction avec les conditions d'environnement. Nous discutons ces resultats.

Individual age of 193 Triturus marmoratus has been assessed by skeletochronology using cross sections of humeral shafts. The sample comes from five breeding localities at various elevations in the Peneda-Geres National Park (North Portugal). Our data confirm that the biology of this species and notably its longevity which can reach at least 15 years in natura in north Portugal, is highly linked to local climate conditions. Histological analysis of skeletal growth marks demonstrates that the older individuals lived in the higher stations. Histological data also allow an assessment of age at the first breeding season which in this sample is most commonly at five years. Finally we offer an explanation for the numerous dividing growth lines most often found in the high altitude specimens as a record of a second slow growing period which at high elevation occurs each year during summer.

The skeletal marks present in numerous po'ikilo-therm Vertebrates are, for numerous workers, the expression of an annual growth periodicity. They have been used, therefore, to calculate the individual age. Nevertheless, few works exist about the nature of these marks. We have begun, by various techniques to study the histo-logical structure of these marks on a series of native Vipera aspis (L.). Skeletal marks are made of two types of osseous tissues: (1) lamellar bone, with parallel fibers, for the annuli, considered to be the result of a period of slow growth; (2) woven fibered bone, for the zones, considered to be the expression of a period of rapid growth. The value of these skeletal marks as an index of age is still to be demonstrated by experimental methods. Resumee Les marques squelettiques presentes chez de nombreux Vertebres poikilothemes, temoignent pour les auteurs d'une periodicite annuelle de la croissance. Elks ont de ce fait ete utilisees dans le calcul de 1'âge individuel. Cependant peu de travaux nous renseignent sur la nature de ces formations. Nous avons entrepris sur un lot de Vipera aspis (L.) indigenes et par des techniques variees, l'etude de la structure histologique de ces stries de croissance. Celles-ci correspondent A deux types de tissus osseux: (1) os lamellaire ou L fibres paralleles, pour les annuli, considere comme le resultat d'une phase de croissance ralentie; (2) os A fibres entrecroisees, pour les zones, consider6 comme le resultat d'une phase de croissance rapide. La valeur des marques squelettiques comme critere d'Lge reste cependant A demontrer par la methode experimentale.

Abstract The age of 103 specimens from a wild population of Cophosaurus texanus (Sauria, Iguanidae) from Mexico is determined by counting skeletal growth rings in the diaphysis of the humerus and phalanges. The skeletochronological analysis of 8 lizards ofknown age supports the practical value of the method. However, inacuracies do occur in practice for age assessement of a few specimens. I n the sample studied, we observed three main age groups (and possibly a fourth one based on a single specimen four years old). The one year age class is the most numerous, accounting for 75 % of the sample studied. Females lay eggs twice each summer with an interval of sixty days between each spawning. The use ofthe skeletochronological method also allows to sort out the individuals belonging to the first or to the second clutch, at every stage of their subsequent lives.

From a comparative viewpoint, this paper deals with structural and functional aspects of skeletal annulations in lizards, especially those displayed by sections cut transversally through the larger bones of the appendages. Cross-sections of the diaphyses exhibit an alternation of concentrically arranged thinner and thicker annulations which were formed during colder and warmer seasons, respectively. The thinner annulations are chromophile and anisotropic but not hypermineralized; they are generally referred to as “resting lines” or “adhesion lines” and are due to a slowing down of the temperature-dependent growth process; they also differ structurally from “cementing reversal lines”. In certain cases, the thinner annulations (here called LAC) are surrounded by bone lamellae and thus compare closely with the annuli on the flat bones. From the study of two lizards of known age, as well as on the basis of a preliminary experimentation with fluorochrom labelling, it has been demonstrated conclusively that the recurrence of the LAC in lacertilians shows an annual regularity. Therefore, it would seem that the LAC can be employed for age determination in lizards.

Histology and cytology of dermal scales of the gymnophionans Ichthyophis kohtaoensis and Hypogeophis rostratus reveal their structure and the nature of their mineralization. Dermal scales are small flat disks set in pockets in the transverse ridges of the skin. Each pocket contains several scales of various sizes. A ring of "hypomineralization" of varying diameter may occur on scales of a particular dermal pocket but bears no relation to the diameter of these scales. Three different layers form the scales and are seen on sections perpendicular to the surface. The cells of the basal layer lie deepest. Each of the two or three more superficial fibrous layers is composed of bundles of fibres that are oriented in parallel. The orientation varies among layers. The striation of the fiber scales has a periodicity comparable to that of the surrounding dermal fibers. Squamulae form a discontinuous layer on the scale surface and are the only mineralized part of the scale. The minerals are deposited both on the collagen fibers passing from the fibrous layers into the squamulae, and in the interfibrillar spaces. Spherical concretions, either isolated or coalescent, reaching up to 1 μm, are found on the surface of the squamulae. The dermal scales of Gymnophiona present some analogies with those of evolved bony fishes. Their characteristics could make them an original model for the study of mineralization.