The problem of age estimation in amphibians and reptiles with annual fluctuations of growth pattern has been considered to be mostly solved after skeletochronological method was introduced back in the middle of 20 century [1]. This method is based on counting the number of registration markers (RM) or lines of arrested growth (LAG), i.e. layers with different optical density within the tubular bones. To date, this method is widely used to estimate age in amphibians [2–6] and reptiles [7–9]. Femoral or humerus bones are normally used as the material for analysis [10], as they are relatively of a large size and contain obvious growing layers. The drawback of this method is that it requires animal euthanasia and, consequently, cannot be considered as humane.
Relying on longitudinal monitoring, ecological and behavioural studies suggest that toe-clipping and distal finger phalanx analysis should be used to collect the material [11–15]. These methods allow the researchers to conduct a life-long study to estimate the growth pattern of individuals. Moreover, this offers a possibility to double-check the obtained results by comparing growth patterns of an individual with the number of the layers within its bone tissue.
It should be noted, though, that in small animals the wintering rings stop occurring after maturation, so when it comes to small lizards it is practically impossible to distinguish between the individuals of six or seven years from much older individuals [15] (Saint Girons et al., 1989). Another problem, which the researchers often face, is that intermediate layers, which can emerge during second annual inactivity – for example, aestivation during summer period in desert or montane species [16, 17] – may hamper the accurate counting of layers [18]. Finally, assessing the exact number of layers may be generally problematic prompting the researchers to use the double-blind method to verify the obtained results [19]. Hence, additional reference data are obviously required to support the results obtained by a skeletochronological method.
In the course of our studies we have noticed that dermal structures on the dorsal part of the claw sheath is multilayered, and suggested that these layers could be useful for age estimation in lizards. We put forward that the proposed procedure might be an effective supplementary method accompanying skeletochronological method, as it may allow using two different structures placed on the same finger to double-check the esteemed age.
To test this hypothesis, we compared the number of RMs (wintering rings) in bone tissue of the finger phalanx with the number of unguis formations of the claw sheath of the same finger in several species of rock lizards belonging to Darevskia genus. These lizards inhabit the Caucasus mountains and hibernate under the harsh and cold conditions during a prolonged snowy winter [20]. The species of this genus have undergone skeletochronological analysis in the works of different researchers, which proves the method efficient and reliable. The results obtained by means of this method demonstrate that these small lizards with snout-vent length of about 50–70 mm live over ten years [9, 13, 15, 21, 22].
The tubule bone structure in reptiles includes internal endosteum and external periosteum areas. The endosteum is partly destroyed by osteoclasts and not suitable for the age estimation [17]. The double line in the bone of the phalanx separates periosteum and endosteum and it is called a “juvenile ring” or resorption line [18] (Fig. 1a). Thus, age estimation in reptiles is based on counting the arrested growth rings or the lines of arrested growth within the periosteum bone closer to the center of diaphysis [17].
Mammalian claws include soft internal structures covered by a rigid horn above [23]. The external keratin horn might include the periodically banded structures; this fact proves the presence of annual differences in physiological condition of the animal [24]. The reptile claw comprises bone of distal phalanx and epidermal sheath, with keratinized corneus of the unguis an less keratinized subunguis epidermis [25, 26]. Surface cornification of scales, unguis and subunguis of reptiles is driven by the expression of beta-keratins of epidermis [27]. Growth process in reptiles is often uneven, which is represented in presence of distinguished layers of cornified corneus [27] and suggested to be a good marker of annual growth lines (Fig. 1b).