The medullary region is entirely filled with a calcified cartilaginous core throughout the ontogeny of all pachypleurosaurids. In ontogenetically young pachypleurosaurids, primary endosteal bony infillings of the vascular canals and a few secondary trabeculae are preserved in the calcified cartilaginous core. Throughout ontogeny, secondary endosteal bony deposits progressively increase in all pachypleurosaurids (i.e. increased remodelling). Accordingly, the vascular canals are secondarily widened by resorption of the primary infillings, as well as of parts of the surrounding calcified cartilage. Subsequently, secondary deposition takes place (Fig. 2). These secondary endosteal infillings of vascular canals are similar to secondary osteons, which are usually found in bone tissue (Francillon-Vieillot et al., 1990), but are here surrounded by the calcified cartilage matrix. These remodelling processes (i.e. secondary endosteal infillings of the vascular canals and secondary trabeculae) are found only in the diaphyseal regions of the long bones in younger individuals, but progressively increase in abundance towards the epiphyseal regions in older specimens.

In both pachypleurosaurid genera, the LAGs are distinct in contrast to the growth zones and annuli, which in some cases change in thickness throughout the ontogeny of an individual (Fig. 3E–H, Fig. 4E–F). All the pachypleurosaurids show minor or no remodelling of the innermost wall of the cortex. The highest grades of remodelling occur within N. pusillus and N. peyeri, but even in these species only parts of the innermost periosteal bone layer are affected and therefore, the growth record can be entirely reconstructed.

The studied pachypleurosaurids show two bone tissue types in the cortex of their long bones; each is differently vascularised, contains a different quantity and shape of osteocyte lacunae, as well as shows a different birefringence based on the alignment of the crystallites (Glimcher and Muir, 1984). The innermost bone layers are composed of a mixture of woven-fibered bone tissue and parallel-fibered bone tissue (wp; Fig. 2, Fig. 3 and Fig. 4), whereas a lamellar-zonal bone tissue type is found in the outer, more peripheral part of the cortex. The innermost bone layer (wp) is visible as an opaque zone in polarised light showing a high quantity of round osteocyte lacunae (Fig. 3D).

The stylopodial elements of both the forelimbs and hind limbs of pachypleurosaurids show constant and high compactness, as shown by compactness profiles (Table 2). The zeugopodial elements of all pachypleurosaurids, in contrast, show more and also larger erosion cavities in the medullary region. The amount of vascularisation appears to increase with stratigraphic age with N. edwardsii showing long bones with the highest degree of vascularisation (Fig. 5C–D), as is also indicated by the compactness indices (Table 2). The humerus, radius, ulna and femur show a statistically significant difference in the mean compactness between N. edwardsii and S. mirigiolensis (paired t-test, t = 2.78; P < 0.05) with the latter exhibiting slightly higher mean compactness values.

The four pachypleurosaurids differ between each other by the grade of the remodelling of the innermost periosteal bone layers of the cortex and the calcified cartilaginous core in the medullary region. They also show variation in the grade of the vascularisation of the cortex, the spacing pattern of the growth cycles, the age when sexual maturity is reached, as well as in their longevities.

S. mirigiolensis specimens show highly compact limb bones except for the ulna (Table 2), which is the only limb bone exhibiting a relatively thin cortex and a larger medullary region.

The innermost region of the cortex is composed of a bone layer of wp (Fig. 3C–D), which is visible as an opaque zone in polarised light and characterised by a high quantity of round osteocyte lacunae (Fig. 3D).

Throughout ontogeny, the calcified cartilage often remains the main component in the medullary region of the long bones. Large specimens show more osseous deposits in the diaphyseal cartilaginous matrix and less towards the epiphyseal area of the medullary region. In a few large specimens, the calcified cartilaginous core is entirely replaced by secondary bone trabeculae in the diaphysis. The calcified cartilage of the medullary region generally shows a few simple vascular canals which are in some cases secondarily widened and filled with lamellar bone. On average, vascular canals of both the medullary region and cortex comprise 7% of the section area (Table 2).

The long bones reveal large parts of, or even a complete, distinct layer of endosteal lamellar bone (sb) that marks the separation between the medullary and cortical region (Fig. 3C, D) and is preserved along its entire border also at the meta- and epiphyseal regions (Fig. 6).

In S. mirigiolensis, the onset of sexual maturity is indicated by several microstructural changes within the corresponding growth cycles: an abrupt decrease in the growth cycle thickness (Fig. 3E–F), a change in the vascularisation pattern (Fig. 5A–B), a change in thickness of the growth zones and annuli (Fig. 3E–H; see light and extinction pattern). Based on those changes, the attainment of sexual maturity is indicated between the second to fourth year in S. mirigiolensis specimens (Table 1; Fig. 3C–F). The largest specimen (T4510) is also the ontogenetically oldest one with 13 LAGs, therefore being in its 14th year at death. It reached sexual maturity during its second to third year (Fig. 3). T4510 developed an external fundamental system (EFS after Horner et al., 2001; or outer circumferential layer, [OCL] after Chinsamy-Turan, 2005) from the age of 10 years onward. The EFS is marked by closely spaced LAGs and is composed of highly organised lamellar bone.

Generally, no or only a few primary osteons and no secondary osteons are found in the cortex of adult specimens. Before sexual maturity is reached, the long bones show several simple radial, and several simple longitudinal vascular canals (Fig. 3A–D; Fig. 5A–B). After sexual maturity, scattered primary osteons are displayed as well. On average of all the limb bones, the cortex is 5% less vascularised after the attainment of sexual maturity than before this event (average observed compactness before sexual maturity: 0.92).

In polarised light, the limb bones sometimes (e.g., T4510) reveal a distinct light and extinction pattern based on the different characteristics of the growth zones and the annuli within each growth cycle after the wp layers and before EFS. Although both growth zones and annuli are comprised of lamellar bone tissue, they can be differentiated by their thickness, by the alignment of the organisation of the crystallites which is reflected by the birefringence (Chinsamy-Turan, 2005 and Glimcher and Muir, 1984), as well as by the quantity and shape of osteocyte lacunae (Francillon-Vieillot et al., 1990; Fig. 3E–H). Before the attainment of sexual maturity, the growth zones are thicker, exhibit more and rounder osteocyte lacunae and are less birefringent in polarised light, whereas the annuli are narrower and reveal a decreased number of flattened osteocyte lacunae, as well as higher birefringence (Fig. 3E–F). After sexual maturity, the growth zones become narrower, contain a lower number of flattened osteocyte lacunae and more equally directed crystallites, whereas the annuli are thicker with more rounder osteocyte lacunae and less birefringent. This distinct pattern continues until the EFS is reached. However, the resulting light and extinction pattern in polarised light is often poorly visible due to the strong coloration of the bone due to diagenesis.

Throughout ontogeny, the calcified cartilaginous core in the medullary region of the long bones of N. pusillus and N. peyeri is only partly replaced by secondary endosteal osseous infillings (Fig. 2). Only the medullary region in the diaphysis of the humerus in large specimens becomes entirely filled with secondary trabeculae (Sander, 1990).

Sander (1990) stated that N. pusillus and N. peyeri reached sexual maturity at the age of three to four. Upon reaching sexual maturity, the growth cycles abruptly decrease in thickness. The subsequent LAGs of the third and subsequent growth cycles gradually converge before the EFS, showing an asymptotic growth curve after sexual maturity (Fig. 2; Sander, 1990). The largest specimens of N. pusillus studied by Sander (1990) show seven LAGs (Fig. 2) and the age of N. peyeri is nine to ten years (Sander, 1990).

Some long bones (humerus, tibia and fibula) of the stratigraphically youngest pachypleurosaurid, N. edwardsii, entirely replace the calcified cartilaginous core of the diaphyseal, metaphyseal and even parts of the epiphyseal regions with secondary trabeculae and few secondary infillings of the vascular canals. In contrast, the radius, ulna and femur always show remnants of the calcified cartilage core of various sizes in the diaphyseal, metaphyseal and epiphyseal regions (Fig. 6A–H), closely resembling the composition of the long bones of the other pachypleurosaurids. Longitudinally oriented simple vascular canals (slvc) and secondary infillings of the vascular canals dominate the medullary region (when not entirely replaced by secondary trabeculae in large specimens; Fig. 5C–D). The long bones of N. edwardsii reveal large parts of, or even a complete distinct layer of endosteal lamellar bone (Fig. 4 and Fig. 6) between the cortex and the medullary region. All the long bones show one innermost bone layer of wp (Fig. 4E–H).

The preaxial elements of both the forelimbs and hind limbs are slightly more compact than the corresponding postaxial elements, but show no statistically significant difference in the mean compactness values and therefore, the null hypothesis cannot be rejected (paired t-test, t = 4.3; P > 0.05). In the limb bones of large N. edwardsii, erosion cavities are more numerous in the medullary region than in the equivalent region of the other pachypleurosaurids studied.

The attainment of sexual maturity is marked by the increased presence of double lines (sensu Castanet et al., 1993) or even more LAGs in the corresponding growth cycle (Fig. 4E–F). Accordingly, a change in the vascularisation (Fig. 4C–F; Fig. 5C–D), such as a higher number of srvc (simple reticular to radial vascular canals), a change in the light and extinction pattern (Fig. 4E–F), but no decrease in growth cycle thickness accompanies the onset of sexual maturity (Fig. 4C–F), contrary to what is observed in the vast majority of extant taxa. Before sexual maturity, the cortex houses a few primary osteons, several simple radial and longitudinal vascular canals (except for the humerus, Fig. 5C). After this event, shorter srvc and a few primary osteons are found in the cortex. Overall, the vascular canals of the cortex are more regularly distributed in N. edwardsii than in the other Neusticosaurus taxa and S. mirigiolensis (Fig. 2, Fig. 3, Fig. 4 and Fig. 5).

N. edwardsii appears to have reached sexual maturity between its fourth and seventh year (Table 1; Fig. 4C–F). The largest studied specimen of N. edwardsii (T3437) shows 22 growth cycles, indicating an age of 23 years (Fig. 4C–D). Sexual maturity is attained during its sixth to seventh year (Table 1; Fig. 4). The thickness of all growth cycles remains constant until the fifteenth growth cycle, time at which an EFS developed. Therefore, the EFS is the only event during the life time of N. edwardsii causing closely spaced LAGs.

The eusauropterygian Nothosaurus from Winterswijk, for example, also shows the plesiomorphic lamellar-zonal bone tissue type, but with a varying spacing pattern of growth cycles (Klein, 2010). Basal pistosauroids often show constant growth cycle thicknesses up to a certain age, but, in contrast to the pachypleurosaurids sampled, also generally exhibit phases of less organised fibrolamellar layers between the parallel-fibered growth cycles (Klein, 2010). The lamellar-zonal bone tissue type generally results from a lower bone deposition rate, which indirectly reflects the metabolism of the animal (Castanet, 2006 and Castanet et al., 1993). The frequent increase in the growth rate (i.e., presence of fibrolamellar bone tissue) might be linked to an increased metabolism, but could also be simply size-related (Cubo et al., 2005), which probably enabled at least the (basal) pistosauroids to spread further through the colder seas as they were the only sauropterygians not restricted to the Tethys (Klein, 2010 and Rieppel, 2000).

Lepidosaurs are the hypothesised living sister group of the Sauropterygia (Hill, 2005 and Müller, 2003). The spacing pattern of the growth cycles of the pachypleurosaurids can be compared to that of Recent lacertilian squamates because they also show the lamellar-zonal bone tissue type (Castanet et al., 1993 and Francillon-Vieillot et al., 1990). N. pusillus, N. peyeri and Recent terrestrial squamates show a similar spacing pattern of the growth cycles (Castanet and Baez, 1991 and Castanet and Naulleau, 1985) and therefore, a similar life history (including the onset of sexual maturity) is inferred. The growth cycle thicknesses of N. edwardsii are the only ones that remain constant throughout the cortex until the EFS is developed; the condition closely resembling the one found in the marine iguanas, Amblyrhynchus cristatus, from the Galapagos islands (Hugi and Sánchez-Villagra, unpublished). These animals are the only living lacertilian squamates that forage exclusively in the sea (Trillmich and Trillmich, 1986). A comparison to the spacing pattern of terrestrial lizards shows that the distinct spacing pattern of constant growth cycle thicknesses of A. cristatus most possibly reflects secondary adaptation to a life in water, since it is not found in any of its terrestrial relatives (Hugi and Sánchez-Villagra, unpublished). In A. cristatus, sexual maturity is histologically indicated by the higher presence of supernumerary LAGs as well as a change in the light and extinction pattern, but neither by an abrupt decrease of growth cycle thickness nor by a change in the bone tissue type (i.e. lamellar-zonal bone tissue type is present throughout the cortex; Hugi and Sánchez-Villagra, unpublished).

Ecological data shows that this histological change occurs at the time when sexual maturity is reached (Trillmich and Trillmich, 1984). According to these authors, the onset of sexual maturity in marine iguanas is reflected in a change of the behaviour in both females and males. Males start to show strong territorial behaviour including male competition to monopolise the access to females. Females, on the other hand, aggregate once a year to lay their eggs on beach sites, which are geographically separated from the normal colony sites of all marine iguanas. In both sexes, these behavioural changes start with the onset of sexual maturity (Trillmich and Trillmich, 1984). In accordance with these observations, we conclude, that sexual maturity in N. edwardsii was reached well before the EFS developed. According to our interpretations the cortex of all the limb bones of N. edwardsii is about 7% less vascularised after the onset of sexual maturity, than before this event (average observed bone compactness before sexual maturity: 0.86).

In A. cristatus the histological changes, especially the light and extinction pattern, described above might reflect the annual reproduction cycle in this species. This hypothesis is supported by slight differences of this pattern between male and female specimens, as well as by the timing when the pattern changes (Hugi and Sánchez-Villagra, unpublished). The change of the growth zones and the annuli of A. cristatus may result from the changing availability of food, climate and the associated mating behaviour, which is sex-dependant (Trillmich and Trillmich, 1984). In pachypleurosaurids, this light and extinction pattern is indicated in S. mirigiolensis and in N. edwardsii. It is either not found in the other pachypleurosaurids or simply obscured due to the strong diagenetic coloration of the bones. S. mirigiolensis additionally shows a mixture of the spacing pattern between N. pusillus and N. peyeri on the one hand, and the stratigraphically youngest pachypleurosaurid N. edwardsii on the other. The prolonged phase of bone deposition after the onset of sexual maturity might be a result of the secondary aquatic adaptation in these rather small reptiles.

Shine and Charnov (1992) studied the growth rates in Recent squamates and showed that large forms tend to mature well before maximum body size is reached, which appears to be the plesiomorphic reptilian condition (Castanet et al., 1988 and Sander, 2000). Phylogenetic analysis confirmed that the evolutionary increase in maximum body size is accompanied by a decrease in the relative size at sexual maturity (Andrews, 1982 and Shine and Charnov, 1992). In summary, large squamates generally display a constant decrease in growth rate after sexual maturity is reached, whereas smaller ones terminate growth at sexual maturity or shortly after this event (Andrews, 1982 and Shine and Charnov, 1992). Changes in growth rate are generally reflected in the bone deposition rate and therefore, in the histology of an individual (Castanet, 1985, Horner et al., 2000 and Padian et al., 2001). Small lizard taxa often display an EFS, which is developed earlier during ontogenesis and which occurs soon after sexual maturity (Ortega-Rubio et al., 1993 and Zug and Rand, 1987). The early development of the EFS exactly reflects the results of the growth analysis by Andrews (1982) and Shine and Charnov (1992). Large reptiles, in contrast, frequently increase their growth cycle thickness slightly again after this event for one or more growth cycles. The subsequent LAGs then align slowly and continuously closer until an EFS is developed (Castanet et al., 1988, Chinsamy et al., 1995, Chinsamy-Turan, 2005 and Erickson, 2005; Hugi and Sánchez-Villagra, unpublished; Saint Girons et al., 1989). However, in both cases, the first abrupt decrease in growth cycle thickness was verified as the time when sexual maturity is reached. This first abrupt decrease in growth cycle thickness occurs in size class B in S. mirigiolensis (Table 1) and in size class F in N. pusillus and N. peyeri (Sander, 1990). This histological change corresponds to the first appearance of sexual dimorphism in these taxa (Rieppel, 1989, Sander, 1989 and Sander, 1990). The development of the EFS only occurs in larger size classes and therefore, does not coincide with the timing when sexual dimorphism is expressed for the first time (Fig. 2: T4178: size class I). The distinction between the two sexes in S. mirigiolensis, N. pusillus and N. peyeri are clearly expressed for example by the ratio of the maximum distal width of the humerus to the minimum width of the humerus (i.e., mid shaft width) (Sander, 1989). In Rieppel (1989) the same data were given, but the ratios were inverted, i.e., minimum width to maximum distal width of the humerus. This ratio, recorded for all four pachypleurosaurids, shows two morphotypes (summary given in Sander (1989): page 631, table 9), interpreted as sexual dimorphism between sex x and sex y. Measurements of N. edwardsii established from Carroll and Gaskill (1985) reveal a first occurrence of the sexual dimorphism at size class B. The histological data for the attainment of sexual maturity coincides with the timing of the first appearance of the sexual size dimorphism (Table 1). In contrast, the development of an EFS only occurs in larger specimens (Table 1) and therefore, does not indicate sexual maturity.