Discrepancies in the number of lines of arrested growth (LAG) in the tissues of the humerus and phalanx of sea turtles

Osteohistology, the study of bone microstructure, provides an important avenue for assessing extinct and extant vertebrate growth and life history. Cortical vascularity and collagen fibre organization are direct reflections of growth rate, while bone growth marks are indicative of absolute age. However, each skeletal element has its own ontogenetic trajectory and microstructure of certain bones may not be a true representation of whole body growth. Extensive comparative study of modern taxa is required to resolve intraskeletal discrepancies among age, vascularity and tissue organization in extinct vertebrates. Despite their comparative utility, studies of bone microstructure in modern taxa are severely lacking. Here, we add to a growing comparative osteohistological database by describing (1) bone tissue organization, (2) growth mark count, (3) sexually dimorphic bone (e.g. medullary bone) and (4) secondary cortical reconstruction in the bone microstructure of a 14-year-old male and 5-year-old female North Island Brown Kiwi (Apteryx mantelli). Transverse and longitudinal histological ground sections were processed and described for femora, tibiotarsi, tarsometatarsi, humeri, ulnae and radii in both kiwis. Cortical bone can generally be described as parallel-fibered tissue, interrupted by cyclical growth marks, with vascular canals oriented longitudinally within primary and secondary osteons. Tissue morphologically resembling medullary bone is present in the hindlimbs of the female, and coarse compacted cancellous bone (CCCB) is found sporadically in the male and female hindlimbs. Lines of arrested growth (LAGs) are present in all hindlimb bones of both kiwi, but remodelling has obliterated all LAGs in the male ulnae and radii. LAG count varies intraskeletally, but large weight bearing elements such as femora and tibiotarsi have less remodelling and, thus, higher number of LAGs. LAG count did not match absolute age in any skeletal element; a maximum of seven LAGs are present in the male kiwi and a maximum of seven LAGs in the female kiwi. The tissue organization within the forelimbs and hindlimbs is reflective of the protracted growth strategy of the North Island Brown Kiwi and congruent with previous studies of the kiwi. LAGs were highly variable throughout the skeleton of the kiwi and a decoupling of age and LAG deposition is apparent from the male kiwi samples. Excess LAGs in the 5-year-old female kiwi may be a product of hatching, egg laying or captivity. Regardless, LAG count variation in the kiwi stresses the importance of intraskeletal sampling when assessing growth patterns of extinct taxa. An extensive ontogenetic sampling of kiwi is necessary for future investigations of bone growth patterns, CCCB formation, medullary bone and LAG deposition and obliteration in these elusive birds.

Somatic growth rate data for wild sea turtles can provide insight into life‐stage durations, time to maturation, and total lifespan. When appropriately validated, the technique of skeletochronology allows prior growth rates of sea turtles to be calculated with considerably less time and labor than required by mark‐–recapture studies. We applied skeletochronology to 10 dead, stranded green turtles Chelonia mydas that had previously been measured, tagged, and injected with OTC (oxytetracycline) during mark–recapture studies in Hawaii for validating skeletochronological analysis. We tested the validity of back‐calculating carapace lengths (CLs) from diameters of LAGs (lines of arrested growth), which mark the outer boundaries of individual skeletal growth increments. This validation was achieved by comparing CLs estimated from measurements of the LAG proposed to have been deposited closest to the time of tagging to actual CLs measured at the time of tagging. Measureable OTC‐mark diameters in five turtles also allowed us to investigate the time of year when LAGs are deposited. We found no significant difference between CLs measured at tagging and those estimated through skeletochronology, which supports calculation of somatic growth rates by taking the difference between CLs estimated from successive LAG diameters in humerus bones for this species. Back‐calculated CLs associated with the OTC mark and growth mark deposited closest to tagging indicated that annual LAGs are deposited in the spring. The results of this validation study increase confidence in utilization of skeletochronology to rapidly obtain accurate age and growth data for green turtles.

Skeletochronological analysis was used to compare stained and unstained cross sections of humeri from Kemp's ridley (Lepidochelys kempii) and loggerhead (Caretta caretta) sea turtles to determine if the 2 histological techniques yielded an equal number of visible lines of arrested growth (LAGs). Stained sections viewed at high magnification under a compound microscope revealed the presence of closely spaced and splitting LAGs, resulting in a greater number of individual LAG counts for these sections when compared to unstained and stained sections viewed at a lower magnification under a dissecting microscope. Prior studies have shown that some of these closely spaced LAGs are annual, and therefore the inability to detect such marks could result in a downward bias in age estimates.

We investigated lines of arrested growth (LAG) of long bone tissues in a total of 157 salamanders of Hynobius kimurae from Tokyo and Kyoto, Japan. The number of LAGs did not differ between femurs, humeri, and toe phalanges. We found that the first LAG is formed after the first overwintering. The number of LAGs varied from 5–14 (x¯=8.8) in reproductive males and 7–12 (x¯=9.4) in mature females in the Tokyo population, while in the Kyoto population, males and females had 6–20 (x¯=9.1) and 7–17 (x¯=9.9) LAGs, respectively. This suggests that the minimum maturation age in males is five yr in Tokyo and six yr in Kyoto, while females of both populations need at least seven yr. The female-larger sexual size dimorphism, recognized in each population of this species seems to be attributable to a greater growth rate in females after the age of male maturity. Body size growth was better in Kyoto than in Tokyo, with average adult SVL being 3.2–3.6 times and 2.2–2.3 times of SVL of metamorphs, respectively. A...

Age structure, growth and longevity was determined in the common toad, Rhinella arenarum, from a sub - urban pond located in the Pampa plains, central Argentina during two breeding seasons, in 2000 and 2008 by using skeletochronology, which relies on the analysis of the annual lines of arrested growth (LAGs) in bones. Both females and males were captured in 2008, while only males were recorded in 2000. Females were significantly larger than males. Mean population age was 2.4 ± 0.9 years in 2000. In 2008, the difference in age was not significant between the sexes (Males: 3.0 ± 0.7, n = 21; Females: 2.6 ± 0.9, n = 12), neither between males in 2000 and 2008. The longev - ity in males of 2000 was 6 LAGs and exceeded that of males (5 LAGs) and females (4 LAGs) in 2008. Von Bertalanffy curves showed that the growth coefficient in the males of 2000 (K = 2.97 ± 0.47) was almost double that of females (K = 1.21 ± 0.10) and males (K = 1.01 ± 0.14) of 2008. Males and females Rhinella arenarum show different morpho - logical and life history traits and the year of sampling can significantly influence the estimation of the studied param -

Skeletochronology was used to determine the ages of 30 loggerhead sea turtles (Caretta caretta) found in French Mediterranean waters. Histological sections were performed with 30 humeri collected on stranded C. caretta on the coast of Provence and the Gulf of Lion. Lines of arrested growth (LAGs) were used to evaluate age, considering only one period of arrested growth per year. Two successive LAGs delimit an interval that is proportional to the individual’s body growth. In a first step, we created a skeletogram for each individual that identified its LAGs. Secondly, we measured the areas delineating successive intervals to determine individual growth patterns over the years. Two methods were used to deduce sea turtle age from histological sections: the correction factor (CF) method and the growth rate (GR) method, the latter using Von Bertalanffy growth function. Age estimations varied from 4.3 to 24.7 years (mean ± SD = 10.1 ± 4.8) with the CF method and from 6.2 to 25.6 years (mean ± SD = 12.2 ± 5.2) with the GR method, with no significant difference between the methods. The study sample was thus mostly composed of juveniles, which is also the most frequent life stage observed in the study area. The annual curved carapace length growth rate calculated with every LAG interval varied from 1.0 to 6.9 cm/year (mean ± SD = 3.5 ± 1.4, ). High variability in interindividual growth rates were observed, which is of interest in understanding the complex lifecycle of these long-lived marine reptiles.

Simple SummaryAge determination is very important for observing life history traits, evaluating vulnerable life stages, and setting proper management and conservation strategies. Age determination in animals has always been tricky. Among other approaches, skeletochronology, an age determination method that involves counting the lines of arrested growth (LAGs) produced during inactive periods, similar to plant year rings, is well-practiced in many animal groups. However, the applicability of this method remains questioned for many species, including amphibians, because of concerns around the scarcity of information on confirmed numbers of annual LAGs (e.g., once or twice in a year), chances of the disappearance of LAGs over time, and which lines exactly to count. Herein, we tested its applicability to Kaloula borealis, a class II endangered amphibian in South Korea, by rearing juveniles in the laboratory for more than one year and comparing the results with the wild population at Lake Sihwa. This study confirmed the formation of one LAG each year and no disappearance of LAGs over time in this species. Furthermore, we were also able to determine the age structure of this wild population accurately. Hence, our study validates using skeletochronology in this species and recommends it for others that show similar growth patterns.Despite having some limitations, the use of skeletochronology—age determination by counting lines of arrested growth (LAGs)—in amphibians is increasing. The main limitation of using skeletochronology is identifying the innermost visible line (IVL) and counting the exact number of LAGs. Thus, we tested its applicability to Kaloula borealis, a class II endangered amphibian in South Korea. We reared juveniles in the lab to investigate the process of bone formation. This confirmed the development of one LAG each year. Hence, our study validates skeletochronology for the age determination of this species and recommends it for others that show similar growth patterns. Furthermore, the comparison of threshold diameters with the IVL of wild individuals confirmed no LAG1 resorption. The average age of males and females in this population was 2.75 ± 1.05 and 3.64 ± 3 years, respectively. We estimated sexual maturity at 2 years with rapid growth up to that stage in both sexes. We found a female-dominated sexual size dimorphism. This study offers accurate information on the life history traits and age structure of K. borealis that may help to evaluate population dynamics in other areas, identify vulnerable life stages and sites, assess the causes of population decline, and set conservation priorities.

Age estimation through skeletochronology and mark-recapture of free-living Liolaemus leopardinus (Squamata: Liolaemidae) from Chile. Age determination is a crucial component of ecological studies. Researchers have relied on different methods and techniques, for example mark-recapture, body size, and skeletochronology, to assess the age of free-ranging individuals. We used all three methods to estimate the age structure of a population of Liolaemus leopardinus, a highly social and saxicolous lizard species endemic to the temperate region of central Chile. This high-elevation and secretive species is considered threatened and, although efforts have been made to reveal more specifc details about the species’ natural history, crucial details of its biology are still unknown. Our goal was to associate the number of Lines of Arrested Growth (LAGs) to snout–vent length (SVL) and use LAGs as an age estimation proxy on free-ranging individuals. For the skeletochronology analyses, a combination of toe-clips was collected when each subject was frst captured in 2012–2013. SVL for all captured individuals was recorded during two different feld seasons (austral spring to fall of 2011–2012 and 2012–2013). SVL data were also available for 10 individuals initially collected and permanently marked in 2005 (one juvenile and nine adults) and recaptured in 2011–2012. Three of those 10 subjects were captured again in 2012–2013. Our results revealed the formation of LAGs in L. leopardinus and a high degree of bone remodeling in both juveniles and adults. This bone remodeling combined with the high rapprochement in peripheral LAGs on the samples of the oldest lizards suggest that phalangeal bones are not suitable for age determination in this species. On the other hand, our mark-recapture results allowed us to assign individuals to four different age-classes when a subject’s SVL was associated with activity periods and recaptures. Individuals of L. leopardinus are long-lived and their lifespan can exceed a decade. Female lizards become sexually mature at three to four years of age.

A Skeletochronological Study of Growth and Age in Relation to Adult Size in Batrachoseps attenuatus

Age estimation of the juvenile stage of Varanus salvator bivittatus using limb bones, namely the big long tubular bones (femur, tibia, humerus) and the small long tubular bones (fibula, radius, ulna, phalanx), was performed using the skeletochronological method. Samples were from Java Island, Indonesia and provided by the pet exporters. Lines of arrested growth (LAG) were present and the first growth mark was visible on a female with a snout-vent length (SVL) of 18.8 cm, and a male with SVL of 19.3 cm. The process of resorption begins in the juvenile stage, and resorption occurs in all long tubular bones. Inside the big long tubular bones, resorption begins earlier and is more extensive than in the small long tubular bones, and in one individual with SVL 25.0 cm, the endosteal bone had completely eroded in all big tubular bones. Resorption also occurred in the small long tubular bones; however, on one individual with an SVL of 25.0 cm, the remaining endosteal bone was still visible only in fibula bone. Linear regression analysis resulted in a weak correlation, statistically insignificant between the SVL and the diameter of the marrow cavity (d) in the fibula bone, while in other bones the correlations were strong and statistically significant (p < 0.005). Among seven tubular bones, the resorption rate in the fibula bone was the lowest but very high in the phalanx bone. Radial osteon formation has occurred in the smallest individual with SVL 14.4 cm to the biggest one with SVL 25.0 cm, and radial osteon density increases with increased SVL. There was no radial osteon in fibula and phalanx bones in any sampled specimen. In general, there were no significant differences in bone diameter (D), marrow cavity diameter (d) and bone thickness (MP) in all long tubular bones in both male and female individuals, and the density of radial osteon on some tubular bones was influenced by body length, but not by sex.

dant bone matrix and lines of arrested growth (LAGs). Conversely, amphibians and reptiles from tropical and subtropical areas, and particularly those inhabiting rain forests with relatively constant environments, are expected to have either ill-defined or no LAGs, since these species presumably have no or scarce interruptions and hence continuous growth. Alternatively, if these species undergo annual patterns of inactivity, then a seasonal pattern of LAGs should be detectable. However, despite the great number of skeletochronological papers recently published on amphibians (Castanet et al., 1993, 1996; Guarino et al., 1995), no one has ever investigated amphibians from tropical rain forests. The aim of this study was to examine skeletochronology in Mantidactylus microtympanum, an endemic anuran from the Malagasy rain forest, and to determine whether this technique was appropriate for estimating age structure of tropical herpetofauna. Blommers-Schlosser (1993) includes M. microtympanum in the subfamily Mantellinae (Ranidae), which is endemic to Madagascar. This large-sized species (snoutvent length up to 100 mm) inhabits rain-forest streams in the southeast of the Grand'Ile, and it is active and visible mainly at night. Males and females differ in some characters, such as belly convexity, biometric ratios, and peculiar structure of cloaca in the female. Both juveniles and adults have a brownish back, although it is mottled in the former. The large size of the species allowed us to perform skeletochronological analysis on phalanges alone and, therefore to mark and release the individuals on the site of

Skeletochronological analysis was performed on Loggerhead sea turtles (Caretta caretta) stranded on the Italian Adriatic and Mediterranean coasts from July 1999 to October 2002. Only the phalanxes of the forelimbs were analysed. The bones were cut at mid‐diaphyseal level. After decalcification, they were sectioned by cryostat and stained with Erlich's hematoxylin. Phalanx cross‐sections showed well‐defined and grossly concentric lines of arrested growth (LAGs). The number of which was correlated with the curved carapax length (CCL). In order to more accurately estimate the individual age, also the number of LAGs lost due to bone resorption was calculated, using osteometric analysis, and added to the number of visible LAGs. Our study showed a longevity ranging from 9 to 18 years for individuals with CCL between 28 and 49 cm; longevity was often greater than 20 years in individuals with CCL over 50 cm. The oldest individual had 83 cm of CCL and was approximately 32–37 years old.

Stegosaurus has unique plate- and spike-shaped osteoderms. Previous studies have focused on the function of the osteoderms; however, ontogenetic development and maturity of these osteoderms with respect to Stegosaurus body growth are little known. In this study, the bone growth of both the skeleton and osteoderms was assessed using thin sections from small to large-sized individuals. In the small individual, the bone histology shows a fibro-lamellar tissue with a radial and/or reticular vascular network in both a body element and an osteoderm. In the medium-sized individual, the cortex shows a longitudinal vascular network and a few lines of arrested growth (LAG) in the skeleton, whereas the osteoderms still retain fibro-lamellar bone and show a reticular vascular network without LAGs. In large individuals, both body elements and osteoderms have fibro-lamellar tissue and multiple LAGs. However, bone tissues from body elements possess external fundamental systems (EFS) at their periphery, whereas osteoderms from the large individual lack EFS. Four histological stages are observed in both the skeleton and the osteoderms in the growth series of Stegosaurus sections: (1) a fibro-lamellar tissue with a radial and/or reticular vascular network, (2) fibro-lamellar tissue with a longitudinal vascular network, (3) with LAGs, and (4) with EFS. The timing of histological changes of osteoderms was delayed from those of the skeleton. This delay indicates that osteoderms might have maintained faster growth rates than the body elements after the maturity of the skeleton.

Ostriches and emus are among the largest extant birds and are frequently used as modern analogs for the growth dynamics of non-avian theropod dinosaurs. These ratites quickly reach adult size in under 1 year, and as such do not typically exhibit annually deposited growth marks. Growth marks, commonly classified as annuli or lines of arrested growth (LAGs), represent reduced or halted osteogenesis, respectively, and their presence demonstrates varying degrees of developmental plasticity. Growth marks have not yet been reported from ostriches and emus, prompting authors to suggest that they have lost the plasticity required to deposit them. Here we observe the hind limb bone histology of three captive juvenile emus and one captive adult ostrich. Two of the three juvenile emus exhibit typical bone histology but the third emu, a 4.5-month-old juvenile, exhibits a regional arc of avascular tissue, which we interpret as a growth mark. As this mark is not present in the other two emus from the same cohort and it co-occurs with a contralateral broken fibula, we suggest variable biomechanical load as a potential cause. The ostrich exhibits a complete ring of avascular, hypermineralized bone with sparse, flattened osteocyte lacunae. We identify this as an annulus and interpret it as slowing of growth. In the absence of other growth marks and lacking the animal's life history, the timing and cause of this ostrich's reduced growth are unclear. Even so, these findings demonstrate that both taxa retain the ancestral developmental plasticity required to temporarily slow growth. We also discuss the potential challenges of identifying growth marks using incomplete population data sets and partial cortical sampling.

Age structure of populations of four species of endemic Malagasy frogs of the genus Mantella (M. aurantiaca, M. baroni, M. bernhardi, M. madagascariensis) was examined by skeletochronology based on 96 specimens from nine different localities. In more than half of these (57%), no lines of arrested growth (LAGs) were found, and the number of LAGs recognized in the remaining specimens was mostly one, and probably two in three specimens. It is generally considered that each LAG corresponds to one year of life; our results therefore confirm that in Mantella populations almost all specimens are in their first or second year of life.