Exaeretodon argentinus PVSJ 38–2002 (Figs 4A, 4B and 8).

Cynodontia was the last therapsid group to evolve, and includes mammals as extant representatives [102]. By the end of the Triassic the group was globally distributed and had diversified into a wide array of taxa varying in lifestyle, diet, and morphology [103]. Traversodontidae is a clade of herbivorous non-mammaliform cynodonts that thrived during the Middle and Late Triassic [104]. Exaeretodon is among the largest members of this clade [105, 106], and femur PVSJ 38–2002 is from the largest individual to have been sampled histologically to date (Table 1) [89, 105, 107]. Exaeretodon fossils represent ~17% of the tetrapod diversity in the Cancha de Bochas Member of the Ischigualasto Formation [20, 67, 69, 89].

Exaeretodon femur PVSJ 38–2002 exhibits a thick compact cortex surrounding a narrow cancellous spongiosa packed with highly remodeled, broken, bony trabeculae (Figs 4A, 4B and 8A). Within the coarse and cancellous spongiosa large erosional spaces are lined with endosteal lamellar bone indicating cycles of bone erosion and redeposition over the course of ontogeny (Fig 8B and 8C). Bone erosion extends into the perimedullar deep cortex, where a few erosion rooms are developed and sparse secondary osteons overprint some areas of primary bone (Fig 8B and 8C). FLB characterizes primary bone throughout most of the cortex. Primary vasculature within the FLB context is composed of mature, highly infilled, and widely spaced longitudinal and circular canals that interweave within a woven bone matrix (Fig 8D and 8E). The intensive lamellar infilling of these primary vascular spaces imparts a “highly organized” morphology, but close examination under cross-polarized light highlights the presence of thin woven bone components between primary osteons (Fig 8D). This pattern persists in all active growth zones in the cortex. Active appositional growth in the cortex is punctuated by two broad regions of mid-cortical growth marks (Fig 8B). The first occurs in the mid-cortex, superficial to the perimedullar region of compacted bone and bony trabeculae (Fig 8E). This first growth mark includes a narrow region of 3–4 annuli and a LAG in the context of a shift toward more highly organized PFB/LFB and reduced vascularity. This signature is traceable around the circumference of the element (Figs 4B, 4C, 8A, 8B and 8E). A circumferential ring of erosional spaces and incipient secondary osteons occurs between each of the annuli (Fig 8E). FLB deposition resumes following this sustained pause in growth, though vasculature is reduced and dominated by more longitudinal primary osteons. This more superficial zone of active growth is terminated by a second more superficial pause in bone apposition again recorded by 3–4 stacked annuli and a LAG in a LFB matrix (Fig 8A and 8B). After this second deceleration, primary bone growth resumes in a final narrow peripheral growth zone. Vasculature in this region is very sparse, and in some areas the element is avascular (Fig 8F). The periosteal border exhibits stacked LAGs and annuli in the context of avascular LFB forming an EFS that signals the cessation of growth and attainment of maximum size (Fig 8F).

The bone tissues of therapsid clades are relatively well known and generally reveal life histories characterized by highly vascularized FLB [105, 108–116]. Recently Veiga and colleagues [105] analyzed multiple appendicular elements at variable ontogenetic stages for Exaeretodon riograndensis from the Late Triassic of Brazil. Our observations for PVSJ 38–2002, though from an individual a bit larger than that sampled by Veiga and colleagues [105], are consistent with those of the largest femur in their study. In both of these Exaeretodon samples, well-vascularized primary FLB characterizes most of the ontogeny [105], though patterns of vasculature vary throughout the cortex in all sampled individuals. Multiple cyclical pauses in bone deposition are also indicated by annuli and LAG, with decreasing bone apposition between these cyclical LAG approaching the periosteal surface. A gradual shift to more highly organized LFB occurs near the periosteal surface in both specimens. In contrast with the Exaeretodon sample of Veiga and colleagues [105], PVSJ 38–2002 exhibits an EFS.

Hyperodapedon sanjuanensis PVSJ 574 (Figs 4C, 4D and 9).

Rhynchosauria is a group of non-archosauriform archosauromorphs restricted to the Triassic Period. The group includes the first large reptiles to exploit herbivory [90, 117–119]. Rhynchosaurs are among the most common members of Triassic faunas and include taxa with well-preserved ontogenetic series [88–90]. Most studies of rhynchosaur growth have focused on rib histology [17–18, 22, 39, 41], with a few analyses exploring histology among multiple elements and/or ontogenetic series [e.g., 19, 117–119]. The Hyperodapedon femur we sampled (PVSJ 574) is similar in size (Table 1, 21.1 cm) to that of another sampled specimen from India (24.8 cm) [19, 90]. Hyperodapedon fossils represent ~60% of the recovered fossils in the Cancha de Bochas Member of the Ischigualasto Formation [20, 67, 69, 90].

The femur of Hyperodapedon (PVSJ 574) exhibits a thick compact cortex. Antero-posterior compression and breakage makes it difficult to discern the nature of the medullary region, but it appears to be filled with broken remnants of a spongiosa composed of thin spicules of remodeled bony trabeculae (Figs 4C, 4D and 9B). There are a few very sparse but large erosional spaces in the deep cortex, with a few extending into more superficial regions of the mid-cortex (Fig 9B and 9C). All of these erosion rooms indicate the onset of limited remodeling of primary cortical bone in Hyperodapedon. Circumferential cementing lines and centripetal deposition hint at the onset of secondary osteon formation in some of the erosion rooms (Fig 9C), but mature secondary osteons are absent. Primary FLB with abundant interweaving, irregular, and reticular vascular networks of primary osteons dominates the cortex of PVSJ 574 (Fig 9D). Throughout the cortex osteocyte lacunae are miniscule but very dense in the woven bone components of the fibrolamellar complex (Fig 9D).

The active growth recorded by such highly vascularized FLB in the cortex of PVSJ 574 is divided into five zones demarcated by circumferential annuli and/or LAG (Figs 4C, 4D, 9A and 9B) that impart a stratified morphology to the transverse section of PVSJ 574. The most external zone is narrower than the deeper zones, though some mid-cortical zones are thinner than the most peripheral zone. From deep to superficial, subtle changes to bone microstructure also point to a relative reduction in the rate of primary bone osteogenesis within each active growth cycle in later ontogeny (Fig 9B, 9C, 9E and 9F). Variation also records changes to bone depositional pace within each cycle. Each growth cycle begins with a pulse of well-vascularized FLB bone, followed by a transition to less vascularized PFB. Within this area of PFB, annuli are more common, and many growth cycles conclude with an appositional cessation indicated by a LAG. This pattern indicates a gradual slowing of growth followed by a stop, and then a resumption of active apposition as ontogeny continues (Fig 9E). These variations are accompanied by a reduction in primary bone vascular anastomoses and osteocyte lacunae density (Fig 9E and 9F). Following each interval of growth cessation the signal reverses, as LFB/PFB grades into FLB and signals a gradual resumption of faster deposition within the next active growth zone (Fig 9E and 9F). Sometimes the resumption of FLB deposition is abrupt (e.g., Fig 9E), and at other times it can occur more gradually (Fig 9F). The zones decrease in width from deep to superficial, with the most external zones preserving the narrowest zones of primary bone deposition (Fig 9A, 9B and 9F). In the most external portions of PVSJ 574, primary vascular canals are either longitudinal, or form a relatively sparse network of longitudinal and circular vascular canals in a sub-laminar pattern (Fig 9F). PVSJ 574 lacks an EFS, though the external cortical bone documents a transition in vascular density and more closely spaced LAG (Fig 9F). This may indicate growth attenuation as adult size is approached, or may simply represent the “reset” of appositional growth following a cessation as observed in the deeper regions of earlier ontogeny cycles in the deeper cortex. Either way, our data suggest that Hyperodapedon was undergoing a period of significantly reduced appositional growth when it died.

Rhynchosaur bone histology has been described for species of Hyperodapedon from India [19] and Brazil [118]. A few isolated and indeterminate rhynchosaur elements from the Ischigualasto Formation that likely can be attributed to Hyperodapedon have also been briefly described [18, 39]. Though the sampled skeletal elements, ontogenetic stage, and the locations of thin-section vary amongst these samples, a general growth pattern emerges for Hyperodapedon that is consistent with data from our sampled femur. The majority of bone apposition in Hyperodapadon is characterized by early deposition of FLB with a significant woven-fibered component and abundant primary vascularization. These FLB tissues occur within cycles that are clearly demarcated by LAG. Cycles between LAG are broadest deeper in the cortex, narrowing toward the periosteal margin. Near the outer circumference of the sampled elements primary bone deposition generally transitions to PFB and/or LFB dominated by sparse longitudinal primary vascular canals, and no EFS [18, 39]. Other sampled rhynchosaur taxa also generally exhibit more well-developed signatures of secondary remodeling, including both endosteal lamellae forming an IFS and deep cortical secondary osteons [18, 118–119]. This general pattern also occurs in the Brazilian rhynchosaur Teyumbaita sulcognathus [118]. In contrast, the bone tissue of Stenaulorhynchus stockleyi from Tanzania preserves moderately vascularized PFB grading externally to LFB punctuated by regular growth marks, with deep cortical erosion rooms and a few secondary osteons, culminating in an EFS [119].

Proterochampsa barrionuevoi PVSJ 606 (Figs 4E, 4F and 10).

The femur of Proterochampsa (PVSJ 606) exhibits a well-defined open medullary cavity devoid of trabecular bone but lined in some regions by endosteal lamellae forming an IFS (Figs 4E, 4F and 10A–10C) [91]. These lamellae crosscut primary bone deposits in the deepest regions of the cortex, and indicate medullary drift driven by cycles of perimedullar resorption and redeposition (Fig 10C). The cortex is dominated by primary bone, but in the posterolateral regions of the deep cortex a few very sparse erosion rooms and a handful of secondary osteons indicate limited remodeling of primary cortical bone (Fig 10B–10D). Even in these areas primary bone tissue persists throughout the cortex, allowing a full view of histological patterns during ontogeny. Diagenetic alteration makes it somewhat difficult to discern the nature of primary bone mineral organization from deeper to more superficial regions in some areas of the cortex, but circumferential organization of osteocyte lacunae around primary vascular canals are distinctive from the surrounding bone matrix and point to a typical FLB organization (Fig 10D and 10E). Osteocyte lacunae in the woven bone regions of the fibrolamellar complex of PVSJ 606 are generally disorganized, but remain relatively sparse and small (Fig 10D). Radial primary osteons dominate the vascular pattern throughout the cortex, but abundant longitudinal and occasional circular anastomoses also occur (Fig 10D–10F). Mid-cortical growth marks are completely absent in PVSJ 606 (Figs 4E, 4F, 10A and 10B). The element records a sharp change in primary bone depositional pattern near the periosteal surface, where primary FLB transitions to highly organized LFB and circumferential stacked LAG (Fig 10E and 10F). These features are consistent with an EFS. This stark transition could exist because the FLB was first eroded, and the EFS then deposited on an erosional surface. If this were the case, we would expect to observe an erosional line that results in truncated primary osteons, and/or woven tissue fabric at the point where the EFS begins. These features are not observed in our sample (Fig 10A, 10E and 10F). Instead, primary bone histology in Proterochampsa indicates a continuously, rapid early ontogeny bone growth pattern culminating in a rapid late ontogeny shift to slower bone depositional rates approaching maximum size.

Our data for Proterochampsa generally align with those of previous studies of closely related taxa, with a few notable exceptions. Other sampled taxa are also characterized by highly vascularized FLB early in ontogeny that transitioned to LFB over life history [121, 124, 125]. Primary vascular patterns in other sampled femora exhibit mostly longitudinal primary osteons with few anastomoses [121]. Secondary osteons are either rare, as is the case in Proterochampsa, or completely absent in all other sampled taxa [121]. Other taxa share the presence of the IFS with Proterochampsa [121]. In most of these other sampled taxa primary appositional growth is punctuated by mid-cortical growth marks or by mid-cortical transition to PFB. A few other specimens share the transition from highly vascularized FLB to more organized LFB at the periosteal border, but in no other proterochampsian so far described is this transition as stark as it is in Proterochampsa [18, 124]. In contrast with previously studied proterochampsians, Proterochampsa PVSJ 606 records the development of an EFS.

Pseudochampsa ischigualastensis PVSJ 567 (Figs 4G, 4H and 11).

The femur of Pseudochampsa (PVSJ 567) exhibits an open medullary cavity (Figs 4G, 4H, 11A and 11B) [91, 92]. Though the deep cortex is broken, an endosteally derived lamellar IFS that truncates primary bone in some areas of the deep cortex can be identified (Fig 11B and 11C). Cortical erosion rooms and secondary osteons are completely absent from this specimen. Close examination indicates that, with the exception of a few patchy deposits in the deepest cortex (Fig 11C), most of primary cortex is dominated by deposits of PFB and LFB (Fig 11A–11D) organized into at least six distinctive zones, most of which are defined by LAG, but some are characterized instead by a zone of avascularity (e.g., Fig 11B). In spite of the highly organized nature of bone mineral and osteocyte lacunae in PVSJ 567, the element is highly vascularized with a dense network of small, irregular, reticular, circular, and radial canals interweaving through the cortex (Fig 11A–11C). For the most part these vascular canals are embedded directly within the primary bone, and lack a surrounding border of centripetally deposited osteonal tissue (Fig 11D–11F). As such, they cannot be diagnosed as primary osteons, and are instead best characterized as simple vascular canals. Exceptions can be found in a few places in the cortex where some osteonal organization exists around longitudinal primary vascular canals (Fig 11C and 11F). Vascular canals are also more abundant and interweaving deeper in the cortex, and become more simply organized, sporadic, and smaller in more superficial active growth zones (Fig 11D). Growth zone width decreases from the deep cortex toward the periosteal surface. Within these zones, a transition occurs from more highly vascularized PFB at the beginning of each to avascular LFB deep to the deposition of at least one LAG at the superficial margin of each zone. This pattern repeats itself in each cyclical growth zone. The most external zone exhibits circumferential layers of LFB with very tiny densely organized osteocyte lacunae. Primary vasculature persists even at the periosteal border where longitudinal canals dominate, but occasional anastomosis with radial and/or reticular canals persists, as observed in deeper cycles. PVSJ 567 lacks an EFS, and was still actively growing, albeit slowly, when death occurred (Fig 11F).

The more highly organized PFB and LFB tissues that characterize Pseudochampsa femoral sample PVSJ 567 throughout ontogeny starkly contrast with the highly vascularized FLB of other sampled proterochampsians, including Proterochampsa [18, 92, 121, 125, 126]. Instead of primary osteons embedded in a woven bone matrix, vascular canals in PVSJ 606 are generally simple and lack clear evidence of centripetally deposited osteonal bone. These simple vascular canals anastomose, but in the context of PFB and/or LFB. That said, the preponderance of radially-oriented vascularity is similar to what we observed in Proterochampsa (PVSJ 606). In sharp contrast with the continuous (or nearly continuous) ontogenetic growth strategy of other protochampsian samples (especially Proterochampsa), tissue histology in Pseudochampsa indicates that growth in this taxon was punctuated by regular cessations [18, 124], and this points to a distinctive life history for this taxon (or at least this individual) relative to its close relatives.

Sillosuchus longicervix PVSJ 085 (Figs 5A, 5B and 12).

The Sillosuchus femoral sample was taken from the poorly preserved holotype specimen (PVSJ 085). The femur is complete but crushed at proximal and distal ends [113]. Although most regions of the external cortex are brecciated, the diaphysis preserves patches of observable microstructural detail (Figs 5A, 5B and 12). The medullary cavity is crushed, making the nature of the medullary space difficult to ascertain (Fig 12A and 12B). The lack of trabecular components and the preservation of an endosteal lamellar IFS in the deepest cortex indicate that the medullary cavity was probably open (Fig 12B and 12C). A few large erosion rooms are restricted to the deep perimedullar cortex, but secondary osteons appear to be absent (Fig 12B and 12C). Isolated patches of compacted coarse cancellous bone, an endosteally derived tissue formed through the compaction of bony trabeculae, are also present (Fig 12C). Highly vascularized FLB comprises the cortex. Throughout the sample, circular and longitudinal primary osteons interweave in a laminar vascular pattern (Fig 12B, 12D and 12E), even at the most superficial periosteal surfaces (Fig 12F). The sample is devoid of annuli and LAG (Fig 12B and 12D–12F), and the absence of an EFS or any signature of increasing histological organization at the periosteal border supports the hypothesis that this large-bodied Sillosuchus specimen was still actively growing at the time of death (Fig 12F).

Sillosuchus shares a primary cortex dominated by highly vascularized FLB with Poposaurus [137], but exhibits a distinctive laminar vascular pattern that is more similar to the pattern observed in Fasolasuchus [135]. The smaller-bodied Poposaurus also differs from Sillosuchus in that it has a growth pattern interrupted by occasional zones of PFB accompanied by at least seven cyclical LAG [137]. Sillosuchus and Poposaurus differ from Effigia, a smaller-bodied relative in which cortices are dominated by PFB with simple longitudinal and reticular primary osteons vascular canals, with only localized deposits of FLB [132]. Our data indicate that Sillosuchus exhibits no histological evidence for slowed growth at the periosteal surface (e.g., the transition to slower growing PFB and/or LFB, appearance of annuli and/or LAG, and/or reduction in vascular density and anastomosis toward the periosteum), and was thus still actively growing at the time of death. Sillosuchus is unique among sampled pseudosuchians, including other closely related poposaurids and more distantly related taxa like Saurosuchus (see below), because it maintained continuous high rates of bone deposition throughout a significant part of ontogeny.

Saurosuchus galilei PVSJ 047 (Figs 5C, 5D and 13).

The femur (PVSJ 047) of Saurosuchus galilei exhibits a broad open medullary cavity lined by layers of endosteal lamellar bone in an IFS that cross-cut primary bone in the deep cortex (Fig 13A–13C). Signatures of bone remodeling are pervasive in the deep cortex of PVSJ 047, where areas of primary bone are overprinted with wide open erosional bays that are often lined with thin layers of centripetally deposited LFB (Fig 13D). Bone resorption extends into the mid-cortex. Sparse secondary osteons are most pervasive in the deep cortex and extend into the external cortex in some areas. That said, remodeling does not completely obliterate the primary bone signature in most regions of the cortex (Fig 13D–13F). Remodeling is more intensive in the thicker anterior cortex, with more frequent erosion rooms that extend toward the outer cortex, accompanied by denser, more highly interwoven, and more immature primary osteons in an FLB context (Fig 13E). An anterior external muscle scar is reflected in tissue microstructure by an area of higher vascularization and intensive remodeling that extends closer to the external cortex in PVSJ 047 (Figs 5C, 5D and 13A). The highly vascularized woven cortex is perforated by an interweaving network of mature longitudinal and circular primary osteons with occasional radial and reticular anastomoses in a sub-laminar or laminar pattern (Fig 13E). FLB deposition is punctuated by at least six mid-cortical LAG throughout ontogeny; cracks in the mid- and outer cortex follow several of these LAG in the cross-section (Fig 13B and 13E), and some cannot be traced completely around the element. Bone organization within each growth cycle transitions from highly vascularized FLB to more highly organized PFB dominated by sparse longitudinal primary osteons (Fig 13E), followed by deposition of a LAG. Each LAG is followed by an abrupt resumption of laminar FLB deposition in the subsequent cycle, in which a wide lamina of woven bone with disorganized osteocyte lacunae immediately follows LAG formation and signals the recovery of faster, more disorganized FLB deposition (Fig 13E). Growth cycles become thinner toward the periosteal surface of the element. Though the periosteal surface is poorly preserved, where it is visible areas of PFB and LFB with longitudinal primary osteons are common, and likely indicate attenuation of growth as PVSJ 047 approaches skeletal maturity. That said, in spite of the very large size of this specimen, PVSJ 047 lacks an EFS (Fig 13F).

Primary bone tissue histology in Saurosuchus is comparable to that of some other large bodied pseudosuchians (e.g., Sillosuchus, Batrachotomus, Effigia, Postosuchus, Decuriasuchus), especially with regard to the dominance of FLB and secondary remodeling that, at least in some individuals, extends into the mid-cortex [17, 132–136]. Sillosuchus and Saurosuchus both exhibit more frequent anastomosing networks of laminar primary osteons when compared to other pseudosuchians, where cortices tend to be dominated by unidirectional primary osteons that generally lack abundant anastomoses [17, 132–136]. Saurosuchus lacks an EFS, just as in Batrachotomus, Decuriasuchus, indicating that these particularly large-bodied species likely continued to increase body size on their paths to skeletal maturity. Saurosuchus also shares the presence of cyclical mid-cortical growth marks associated with localized areas of PFB with most other sampled pseudosuchians [17, 132–136].

Trialestes romeri PVSJ 368 (Figs 5E, 5F and 14).

Trialestes romeri is one of the earliest known members of non-crocodyliform crocodylomorphs [97, 98]. Its anatomy suggests that it was an active terrestrial carnivore capable of cursorial locomotion [97–99]. Trialestes fossils account for just 0.1% of vertebrate faunal diversity recovered from the Cancha de Bochas Member of the Ischigualasto Formation [20, 67, 69]. Our data for Trialestes are the first for the genus, and add to the previously known histological patterns observed in other basal crocodylomorphs, including the humerus of Terrestrisuchus [17, 18], the femora of Hesperosuchus [5, 17, 18] and Saltoposuchus [138], and the tibia and fibula of an unnamed large bodied South African crocodylomorph [BP/1/8484, 139]. There is no femur associated with the holotype of Trialestes [97], but a referred specimen indicates a maximum femoral length for previously studied Trialestes of just 20.4 cm [97, Martínez, pers. comm]. The Trialestes femur in our sample (PVSJ 368) is associated with a partial skull, axial column, shoulder and pelvic girdles, and forelimb and hindlimb elements that share autapomorphies with the holotype [97, Martínez, pers. comm]. Femur PVSJ 368 is more than twice the length of femora from other known Trialestes specimens [97] (Table 1; 42 cm).

The right femur of Trialestes romeri (PVSJ 368) exhibits an open medullary cavity lined by a thin border of endosteal LFB forming an IFS (Fig 14A–14C). The posterior margin of the element is not preserved. Preserved regions of the cortex lack signatures of primary bone remodeling; erosion rooms and secondary osteons are absent (Fig 14B and 14C). Highly vascularized primary FLB dominates the cortex. Abundant interweaving circular and longitudinal primary osteons form a laminar vascular network (Fig 14D). In some areas occasional radial and reticular anastomoses indicate a sub-plexiform pattern (Fig 14F). The anteromedial mid-cortex documents a zone of reticular vascularity, with abundant radially-oriented primary osteons that may indicate preferential directional growth (Figs 5E, 5F and 14A). In more superficial regions of the cortex primary osteons are mature, and vascular canals are quite narrow (Fig 14E and 14F). In the outermost regions of the cortex the dominant FLB transitions in places to narrow patches of nearly avascular LFB that cannot be traced around the cross-section (Fig 14E). Only sparse longitudinal primary osteons are present in this zone of reduced primary bone deposition (Fig 14F). More superficially, deposition of FLB with laminar vascularity resumes and continues unabated nearly to the external margin of the element. At the periosteal surface, osteocyte lacunae become aligned in parallel rows in a PFB/LFB context, as primary osteons shift to a more longitudinal orientation (Fig 14F). The EFS is absent indicating that though primary bone deposition may have been slowing, Trialestes was still growing when it perished (Fig 14F).

Trialestes femoral histology is most similar to that of the humerus of Terrestrisuchus [17] and the femur of Saltoposuchus [138]. These taxa share the presence of open medullary cavities devoid of cancellous bony trabeculae, but lined with endosteally derived LFB forming an IFS with BP/1/8484 [139], and contrast with the trabeculae-filled medullary cavity observed in Hesperosuchus [5, 17]. In Trialestes, Terrestrisuchus, and Saltoposuchus some regions of the cortex are wider than others, and the cortices are highly vascularized and become more organized toward the periphery. Terrestrisuchus and Saltoposuchus exhibit a predominance of longitudinal primary vascular canals within an FLB context, whereas Trialestes exhibits an interweaving vascular network, potentially indicating a relatively faster bone depositional rate. Where Terrestrisuchus exhibits a single mid-cortical LAG, Saltoposuchus and Trialestes exhibit annuli. Saltoposuchus exhibits two zones of increased primary bone organization, one in the mid-cortex and one near the periosteal surface. In Trialestes a single annulus is situated near the periosteal surface. These data contrast with those for Hesperosuchus [17] and BP/1/8484 [139], in which primary cortices exhibit slower growing PFB and LFB punctuated by more regular LAGs. Interestingly, the only basal crocodylomorph that exhibits significant remodeling by secondary osteons is Hesperosuchus [5, 17]. None of these taxa exhibit a peripheral EFS, though BP/1/8484 exhibits peripheral LAGs that are increasingly more closely spaced [139].

Sanjuansaurus gordilloi (PVSJ 605) (Figs 6A, 6B and 15).

The holotype right femur of Sanjuansaurus gordilloi (PVSJ 605) is brecciated, but details of general primary bone growth patterns and bone microstructure are discernible throughout the cortex (Figs 6A, 6B and 15) [87, 88]. The medullary cavity is open and lined with a few thin avascular endosteally derived lamellae forming an IFS (Fig 15A–15C). Though the element is poorly preserved, erosion rooms, secondary osteons, LAGs, and annuli appear to be absent throughout the cortex where histological structure can be clearly observed (Fig 15C and 15D). Instead, highly vascularized primary FLB forms the entire cortex. Reticular vascularization dominates in the deeper regions of the cortex of PVSJ 605, and primary osteons are mature and highly infilled. Laminar vascularity becomes more common in the mid-cortex, with interweaving circular and longitudinal primary osteons uninterrupted by growth marks (Fig 15D and 15E). Where preserved, the periosteal surface becomes more organized as patches of PFB and longitudinal primary osteons are more common, pointing to attenuation of growth as skeletal maturity is reached (Fig 15F). The EFS is absent in PVSJ 605.

Herrerasaurus ischigualastensis PVSJ 614 (Figs 6C, 6D and 16).

The left femur of Herrerasaurus ischigualastensis (PVSJ 614) is slightly crushed, and some of the bony cortex is displaced into a relatively small but open medullary cavity (Figs 6C, 6D, 16A and 16B). The medullary cavity is lined by distinctive layers of avascular endosteal LFB forming an unusual two-part IFS (Fig 16C). The deepest cortical layer exhibits a few thin internal lamellae with highly organized lamellar bone fiber orientations. Slightly more externally, a single row of wide and open vascular channels is intercalated between these internal lamellae and another group of 5–6 thin highly organized external lamellae (Fig 16C). The interspersed vascular spaces are suggestive of remnant partially eroded and remodeled medullary bone. This region of perimedullar endosteal remodeling cross cuts the deepest preserved region of the cortex (Fig 16C). In the deepest regions of the cortex primary bone tissue is completely obliterated by multiple generations of well-developed secondary osteons forming dense Haversian bone (Fig 16C and 16D). This remodeling doesn’t extend beyond the deep cortex, and secondary osteons are absent in the middle cortex. The primary cortex is dominated by well-developed FLB characterized by dense vascularity in laminar and reticular interweaving networks (Fig 16E). This cortical structure is consistent throughout the cortex, though in the mid-cortex laminar bone tends to become slightly more dominant (Fig 16E). The element is devoid of cortical growth marks. At the periosteal surface vasculature decreases slightly and becomes dominated with immature longitudinal and sparse circular primary osteons. The preserved posterolateral region of the femur exhibits patches of PFB that are either dominated by longitudinal primary osteons or are avascular (Fig 16F). The anterior, medial, and lateral periosteal surfaces, where preserved, are characterized by abundant vasculature and immature primary osteons, indicating ongoing and rapid primary bone apposition in these regions (Fig 16B). The specimen lacks an EFS (Fig 16F).

These results are consistent with those reported by Ricqlès and colleagues [17, 18], specifically in relation to the densely vascularized primary fibrolamellar cortices. In other studied Herrerasaurus specimens vascular networks tend to be plexiform, with an abundance of circular vascular canals instead of the laminar-reticular pattern that dominates femur PVSJ 614. Moreover, thin layers of avascular endosteal bone line the medullary cavity to form an IFS in other sampled specimens, and secondary osteon formation is limited or absent [17, 147]. In contrast, PVSJ 614 exhibits a distinctive pattern of intensive secondary remodeling, with both endosteal lamellar bone forming the IFS and secondary osteons that are dense in the deep cortex. This may point to a somewhat older skeletal age for our sampled specimen relative to other sampled elements, or it may relate to a difference in functional environment (since other sampled specimens included a tibia and a humerus). Our sample includes a thin periosteal zone of avascular PFB/LFB, at least on the posterior surface of the element, that is similar to that observed in other sampled specimens. A similarly sized Herrerasaurus tibia exhibits an EFS, and also lacks cortical growth marks [147], but other sampled specimens lack definitive EFS, and thus were still actively growing when they died.

Eodromaeus murphi PVSJ 561 (Figs 6E, 6F and 17).

We sampled the right femur of the theropod Eodromaeus murphi (PVSJ 561) [83]. The element is only slightly crushed and bone microanatomy is very well-preserved (Figs 6E, 6F, 17A and 17B). The open medullary cavity is somewhat crushed, but endosteally derived avascular lamellar bone rings the regions of the medullary space that persist to form an IFS (Fig 17C). Aside from this evidence of bone erosion and redeposition around the medullary cavity there is no indication of intracortical bone remodeling. There are no resorption cavities, and secondary osteons are absent (Fig 17A–17D). Primary FLB is present throughout the cortex, with mostly primary osteons embedded within a woven bone matrix preserving abundant disorganized osteocyte lacunae (Fig 17C–17E). The density of organized LFB surrounding the many vascular canals imparts a sense of more detailed organization of primary tissue when the specimen is viewed at low magnification (Fig 17B). However, closer study at higher magnification reveals the disorganized nature of the woven component of the fibrolamellar complex (Fig 17C and 17D). Primary osteon orientations vary throughout the cortex of PVSJ 605. In some areas, vascularity is dominated by longitudinal primary osteons, while in other areas they interweave with circular, radial, and reticular osteons that record vascular patterns including reticular, laminar, and sub-plexiform patterns (Fig 17B–17D). In some regions primary osteons are closely packed, leaving only thin interspersed regions of woven bone surrounding them (Fig 17D). There are no LAGs or annuli in the cortex of the femur of Eodromaeus, indicating that primary osteogenesis continued unabated until death in this individual. The periosteal border exhibits transition in bone fiber orientation. In the external cortex, a very thin layer of much more highly organized PFB characterized by osteocyte lacunae arranged in thin parallel laminae is accompanied by a decrease in primary vasculature (Fig 17E and 17F). Vasculature in this final bone depositional interval of femur PVSJ 605 is much more sporadic, with mostly immature longitudinally-oriented primary osteons, including some that are open at the periosteal surface. Femur PVSJ 561 lacks an EFS and points to ongoing growth at the time of death (Fig 17E and 17F).

Eoraptor lunensis (PVSJ 559) (Figs 7A, 7B and 18).

The Eoraptor femoral sample was extracted from a referred right femur recovered 25 cm from the holotypic skeleton [84]. The element exhibits some breakage, but microanatomical details are well-preserved throughout. Femur PVSJ 559 exhibits a wide and open medullary cavity that is partially lined with centripetally deposited layers of endosteal lamellar bone forming an IFS (Fig 18A–18C). The deep cortical histology of this element is highly variable. In some areas the IFS lamellae ringing the medullary space overlap deep cortical deposits of primary fibrolamellar bone (Fig 18C). In other areas the IFS overlaps generations of secondary osteons that reworked an isolated patch of the deep cortex (Fig 18D). The adjacent endosteal lamellae are themselves indicative of extensive remodeling, with large (~550 microns) circumferentially oriented erosion rooms formed at the boundary between endosteal lamellae and older deposits of primary FLB (Fig 18B and 18D). These erosion rooms exhibit thin external layers of centripetally-deposited LFB (Fig 18B). An arc of compacted Haversian tissue persists between these layers of endosteally derived lamellar bone (Fig 18D). The cortex of PVSJ 559 can be divided into three regions that record transitions from more highly vascularized disorganized primary FLB for most of ontogeny to an interval of somewhat more organized lower vascularity PFB for an interval during later mid-ontogeny, and then back to a phase of more active growth in the latest recorded ontogenetic stage (Fig 18E). The deepest primary cortex exhibits highly vascularized primary FLB with circular and longitudinal primary osteons interweaving in a laminar pattern. In some regions this pattern transitions into one with an abundance of radial and more irregularly organized primary osteons, but always in the context of a woven bone matrix that exhibits densely packed osteocyte lacunae consistent with a standard fibrolamellar complex (Fig 18E and 18F). Moving superficially, a transition in primary bone depositional pattern is indicated by a temporary transition in bone mineral and vascular organization. In this region, bone mineral organization is predominantly parallel-fibered, with localized patches of even more highly organized LFB (Fig 18E and 18F), and vasculature is sparse and reduced to simple mature longitudinal primary osteons (Fig 18E and 18F). In the most superficial regions of the cortex, PVSJ 559 documents a return to a more disorganized FLB depositional pattern (Fig 18E and 18F). A sub-plexiform vascular network dominates this region of the cortex, with abundant circular, longitudinal, and radial primary osteons interweaving in a sub-plexiform pattern (Fig 18E). The Eoraptor femur PVSJ 559 lacks LAGs and a peripheral EFS, though the perturbations near the outer cortex are suggestive of the onset of growth rate attenuation (Fig 18F).

Chromogisaurus novasi (PVSJ 845) (Figs 7C, 7D and 19).

We sampled the holotypic partial right femur of Chromogisaurus [81, 82] (Figs 7C, 7D and 19). Bone microstructural data are well-preserved in the mid-diaphysis in spite of breakage at both ends and some overall compression and fracturing of the element. Femur PVSJ 845 exhibits an open medullary cavity devoid of trabecular bone (Fig 19A and 19B). At least four cycles of endosteal remodeling are preserved that include both endosteally-derived LFB as well as secondary osteons (Fig 19B and 19C). The most superficial of these LFB layers likely formed first during an early period of relatively early ontogenetic medullary drift and endosteal bone remodeling that obliterated earlier deposits of deep cortical primary bone tissue (Fig 19B–19D). Following the initial pulse of outwardly focused medullary drift, centripetal deposition of LFB would have resulted in a narrower medullary cavity (Fig 19C and 19D). Some of this endosteal LFB underwent a sequence of additional secondary remodeling as ontogeny proceeded, and was eventually replaced by dense Haversian bone (Fig 19B–19D). A second phase of bone erosion occurred more deeply and adjacent to the medullary cavity, resulting in deposits of LFB that cross-cut these earlier deposits of Haversian bone (Fig 19C). A similar cycle of bone erosion and redeposition occurs at least once more in the perimedullar space, with the formation of a third endosteal lamellar layer that is partially remodeled by secondary osteons (Fig 19C). Here, between the third and fourth cycles, large open erosional spaces are dispersed among secondary osteons and indicate the ongoing process of bone resorption and redeposition (Fig 19B). The fourth, final, and deepest signature of perimedullar remodeling is a bit more unusual (Fig 19B and 19C). Though in some regions the medullary cavity is lined by a fairly typical endosteal IFS (Fig 19C), in at least one area of the deepest cortex a zone of longitudinally vascularized woven fibered bone tissue lies deep to the most internal deposits of endosteal lamellar bone, and abuts a small region of the medullary cavity (Fig 19C). This patch of woven bone tissue deep to endosteal lamellae and lining the medullary cavity in Chromogisaurus exhibits some of the diagnostic features of both medullary bone, a sex-specific bone tissue produced in females during egg-laying [148, 149], as well as a paleopathological condition described for at least one other basal sauropodomorph [150, 151].

At the same time that these cycles of remodeling overprinted signatures of primary tissue in the deep cortex, appositional growth in more superficial areas continued. The primary cortex of PVSJ 845 is characterized by the continuous deposition of FLB vascularized by abundant longitudinal primary osteons with occasional circular anastomoses (Fig 19B and 19D–19F). Most superficially, no major transition in primary bone depositional pattern is indicated, though primary vascularization gradually becomes more uniformly dominated by unidirectional longitudinal primary osteons (Fig 19E and 19F). Intracortical LAGs and a peripheral EFS are absent from femur PVSJ 845 (Fig 19F). Appositional growth was unabated at the time of death for this Chromogisaurus individual.

Growth strategy group I—Slower & cyclical.

Just one taxon resides in GSG I, which is reserved for Ischigualasto vertebrates that exhibit a dominant pattern of histological features indicative of relatively slow growth rates (Fig 21A, Table 2). Future analyses of additional taxa may increase membership within this group. Slow growing and highly organized PFB and LFB characterizes Pseudochampsa and signals an overall slower rate of bone deposition in this taxon when compared to other sampled Ischigualasto taxa. In extant vertebrates these tissues have been experimentally shown to form at apposition rates of 5 μm or less per day [7]. Primary vasculature is abundant, but canals are simple and generally lack centripetal deposition of osteonal bone. Pseudochampsa experienced regular drops in bone depositional rate signaled by changing bone mineral organization from PFB to LFB, coincident with reduced diameter, density, and directionality of primary vasculature as well as the presence of highly organized, tiny, and diminished osteocyte lacunae. These reductions in primary bone deposition occur before and after multiple LAGs indicating regular and recurrent cessation of growth throughout ontogeny. Bone organization surrounding LAGs demonstrates a gradual slowing and cessation, and a similarly gradual resumption of primary osteogenesis. At least 4–5 cycles that narrow from the deeper to more superficial cortex indicate a regimented periodicity and regular attenuation of primary bone apposition over life history for this Pseudochampsa individual. The cortex lacks evidence of bone remodeling in spite of the relatively mature ontogenetic status of this specimen (Fig 20). It is possible that a broken fragment floating within the open medullary cavity indicates that some endosteal perimedullar erosion and redeposition occurred, but this is uncertain. These data are most similar to those recorded in the femoral histology for Chanaresuchus bonapartei from the Los Chañares Formation of Argentina [124]. These distinctive patterns set Pseudochampsa and Chanaresuchus apart from other sampled proterochampsians (see below), as well as other sampled Ischigualasto taxa, and point to a cyclical reduced rate of osteogenesis throughout ontogeny [124].

Growth strategy group III—Fast & continuous.

GSG III includes the proterochampsian Proterochampsa, the poposaurid Sillosuchus, the crocodylomorph Trialestes, and all of the sampled dinosaurs, including Herrerasaurus, Sanjuansaurus, Eodromaeus, Eoraptor, and Chromogisaurus (Fig 21C, Table 2). GSG III taxa share continuous deposition of FLB with abundant anastomoses of dense vascularity regularly approaching or attaining laminar and plexiform patterns during growth history. In contrast with taxa in GSG I and II, members of GSG III lack cyclical growth mark evidence of mid-ontogeny cessation or significant decelerations of appositional growth until later in ontogeny. Though these patterns might seem to be an indication that GSG III merely designates a group of relatively young individuals, comprehensive metrics of skeletal maturity actually point to relatively older ontogenetic status for all GSG III taxa (Fig 20).

Evidence of bone remodeling is present in all GSG III taxa, ranging from simple medullary drift/formation of the IFS to scant intracortical primary bone resorption and/or sparse secondary osteon formation to dense Haversian bone (Fig 20). Trialestes, Sanjuansaurus, Eodromaeus exhibit perimedullar IFS, but lack any evidence of intracortical remodeling (e.g., erosion rooms and/or secondary osteons). Sillosuchus lacks secondary osteons, but exhibits both the IFS and perimedullar and mid-cortical erosion rooms that are the harbingers of ongoing bone resorption. Proterochampsa exhibits an IFS, sparse deep cortical secondary osteons, and mid-cortical erosional bays, all indicative of a high rate of bone metabolism. The dinosaurs Herrerasaurus Eoraptor, and Chromogisaurus exhibit the IFS, intracortical erosion rooms, some deep cortical secondary osteons, and are the only Ischigualasto vertebrates to exhibit intensive remodeling that resulted in the formation of perimedullar/deep cortical dense Haversian bone (Fig 16B and 16C; Fig 18B and 18C; Fig 19B–19D).

Most GSG III taxa are characterized by an external cortical transition to occasional patches of more organized patches of PFB and/or LFB with longitudinal primary vascular canals indicative of slowing primary osteogenesis. These shifts may relate to the approach of skeletal maturity in later ontogeny, but appositional growth was ongoing, albeit at somewhat attenuated rates in most taxa. It is interesting to note that among the GSG III taxa Chomogisaurus does not exhibit a major shift in bone appositional pattern consistent with cessation of growth and skeletal maturity, in spite of the presence of an perimedullar tissue that exhibits a suite of diagnostic features that are more consistent with medullary bone than with pathological bone [148–151]. Medullary tissue is present at the endosteal surface in sexually mature females during egg-formation [148, 149], in constrast with pathological bone, which is deposited at both endosteal and periosteal surfaces [150, 151]. The potential presence of medullary bone can only be confirmed with the sampling of additional elements. If present in the absence of the EFS, it would signal an intriguing decoupling of sexual and somatic maturity in this early sauropodomorph.