Histology and microanatomy of vertebrae, ribs, haemal
arch, and humeri and femora of 10 individuals of Stereosternum and two dorsal ribs of
1 individual of Brazilosaurus were studied. All individuals had achieved a body length
of 50 cm (equal to 65 % of the maximum known body length) or larger. All sampled
bones are highly osteosclerotic due to the reduction of medullary cavities
and the filling of medullary regions by endosteal bone. Calcified cartilage
occurs – if at all – only locally in small clusters in the medullary
regions of midshaft and in higher amounts only in non-midshaft sections of
long bones and towards the medio-distal rib shaft, respectively. The primary
bone tissue consists of highly organized parallel-fibred tissue and/or
lamellar tissue, which is in most samples relatively lightly vascularized or
even avascular. If present, vascular canals are mainly longitudinally
oriented; some show a radial orientation. Simple vascular canals as well as
primary osteons occur. Some of the latter are secondarily altered, i.e. widened. Remodelling of the periosteal cortex is only documented by few
scattered erosion cavities and secondary osteons. The tissue is regularly
stratified by lines of arrested growth (LAGs), which usually appear as double or multiple rest lines,
indicating strong dependence on exogenous and endogenous factors. Because of
the inhibition of periosteal remodelling the growth record is complete and
no inner cycles are lost. Individuals of Stereosternum show a poor correlation of body
size and number of growth marks, which might be the result of developmental
plasticity. Brazilosaurus shows a highly organized, avascular lamellar tissue and a high
number of regularly deposited rest lines throughout the cortex of the ribs.
The medullary region in the ribs of Brazilosaurus is distinctly larger when compared to ribs
of Stereosternum. However, strong osteosclerosis is obvious in both taxa, pointing to a
high degree of aquatic adaption. Ribs of Stereosternum, Brazilosaurus, and Mesosaurus are clearly distinguishable
from each other by the distribution of the periosteal and endosteal
territory. Furthermore, Brazilosaurus differs in its growth pattern (i.e. spacing of rest
lines) when compared to Stereosternum and Mesosaurus.
Introduction
The Permian family Mesosauridae is involved in several key events of the
history of life and that of natural sciences. First of all, mesosaurs are
exclusively found in early Permian black-shale and limestone deposits from
both southeastern South America (the Irati Formation in southern Brazil and
the Melo Formation in Uruguay) and southwestern Africa (the Whitehill
Formation in Namibia and in western South Africa) (Oelofsen and Araújo,
1987). Because of this geographical distribution, which supports the theory that the
two continents were once connected, mesosaurs were among the fossils cited
by Alfred Wegener as a line of evidence to corroborate his theory of
continental drift (Du Toit, 1937). The epicontinental Whitehill-Irati Sea
was home to all three mesosaurid species: Brazilosaurus sanpauloensis Shikama and Ozaki 1966, Mesosaurus tenuidens Gervais
1865, and Stereosternum tumidum Cope 1886. The majority of studies place them among Parareptilia
(e.g. Modesto, 2006; MacDougall et al., 2017, 2018; Tsuji and Müller,
2009), whereas others suggest that mesosaurs may instead represent the basalmost clade of Reptilia sensu Modesto and Anderson (2004) (Laurin and Reisz,
1995; Laurin and Piñeiro, 2017).
In addition to their importance in geology, mesosaurs are also renowned for
being the very first secondarily marine amniotes. Alongside the geological
evidence, their aquatic lifestyle is supported by several anatomical
characteristics: the elongated body, skull, neck and tail; the presence of
pachyostosis in the entire skeleton of Mesosaurus (de Ricqlès and de
Buffrénil, 2001; Modesto, 2010); and their paddle-like autopods. Furthermore, mesosaurs have been suggested to be viviparous (Piñeiro et al.,
2012) and to display humeral microanatomy consistent with a marine lifestyle
(Canoville and Laurin, 2010).
However, the degree to which they were aquatic is controversially discussed,
and whether they might have been partially terrestrial is also still
debated. Morphological evidence suggests limited in-land locomotive
abilities (Modesto, 2006; Núñez Demarco et al., 2018). For some
authors (Romer, 1956; Carroll, 1982; Chiappe and Chinsamy, 1996), the
specialized dentition of mesosaurs was used for filter feeding, while Pretto
et al. (2012) see in the tooth microstructure of Stereosternum evidence for an active
aquatic predatorial behaviour. In their estimation of swimming speed for
Mesosaurus, Villamil et al. (2016) came to the conclusion that this species was a slow
swimmer living in shallow waters, feeding on slow prey, or possibly
filter feeding, rather than actively pursuing rapid prey. To finish,
Núñez Demarco et al. (2018) argued that the axial skeleton of
Mesosaurus displays a semi-aquatic morphometric pattern, suggesting that “maybe only
juveniles and young adults inhabited aquatic environments” while “more
mature individuals might hypothetically have spent time on land”.
Regardless of whether mesosaurs lived constantly in shallow marine
environments or only during certain phases of their life, as they are the
first secondary aquatic tetrapods, the course of their adaptation to living
in water bears important insights into general patterns and processes
triggering this specialization. For this reason, the histology of
Mesosaurus and Stereosternum has been investigated by several authors (Nopcsa and Heidsieck, 1934;
de Ricqlès, 1974; de Ricqlès and Buffrénil, 2001; Canoville and
Laurin, 2010). They found that skeletal elements were affected by two main
mechanisms resulting in a bone mass increase: (1) pachyostosis, which ensues
from the hyperplasia of the periosteal cortex, generally results in the
swollen appearance (hypertrophy) of bones and is particularly well expressed
in the banana-shaped ribs of Mesosaurus; and (2) osteosclerosis, which corresponds
to an increased compactness of the inner bone structure, is achieved through
the condensing of the medullary regions by the retaining of calcified
cartilage and/or by endosteal deposits. The retaining of calcified cartilage
is the result of incomplete endochondral ossification, indicating
paedomorphosis, whereas intensive endosteal deposits follow after complete
substitution of calcified cartilage, indicating that endochondral ossification
is quite complete (de Ricqlès and Buffrénil, 2001). Thus, both of these
processes lead to osteosclerosis, but completely different processes are
involved.
Despite having been the subject of multiple studies, histology in mesosaurs
has always been examined through the prism of adaptation to living in water.
Only a few specimens have been studied firsthand (Nopcsa and Heidsieck,
1934; de Ricqlès, 1974) and then referred to in other studies (de
Ricqlès and Buffrénil, 2001; Canoville and Laurin, 2010). Growth and
ageing patterns of their bones (skeletochronology) were virtually not
investigated, except for one morphological study based on size ratio
comparisons and involving only two specimens (Bickelmann and Tsuji, 2018).
Herein we perform the first skeletochronological study of mesosaur material,
including 11 individuals (mostly representing Stereosternum) of which in part
multiple bones were sampled. This includes the first histological and
microanatomical data for the mesosaur Brazilosaurus and largely emended and expanded
knowledge on Stereosternum, of which previously only ribs were studied.
Institutional abbreviation
BSPG: Bayerische Staatssammlung für Paläontologie und Geologie,
Munich, Germany.
IGPB: Institut für Geowissenschaften, Paläontologie, Universität
Bonn, Bonn, Germany.
MB: Museum für Naturkunde, Leibniz-Institut für Evolutions- und
Biodiversitätsforschung, Berlin, Germany.
SMNK: Staatliches Museum für Naturkunde, Karlsruhe, Germany.
Material and methodsMaterial
Long bones, ribs, a haemal arch, and vertebrae from 10 individuals of
Stereosternum and two ribs of 1 individual of Brazilosaurus were sampled for bone histological study
(Table 1). Little information is available regarding the respective
localities of these specimens aside from the fact that they all originate
from the Irati Formation in southern Brazil. Multiple samples were taken
from each individual (Table 1).
With the exception of the specimen of Brazilosaurus (BSPG 1965 I 131), all specimens were
historically labelled as Mesosaurus brasiliensis but we reidentified them as Stereosternum tumidum based on diagnostic
features such as the number of presacral vertebrae and, if available, the
head/neck proportion (see Araújo, 1976; Oelofsen and Araújo, 1987;
Modesto, 1999). Apart from MB.R.1988, all individuals are fairly complete,
with only individual limb elements or parts of the tail missing. MB.R.5605
is the most complete individual in our sample with an exceptionally complete
tail preserved. This individual and its bone proportions served as a proxy for the
reconstruction of body length estimates for less complete individuals (Table 1). MB.R.1988 is the least complete specimen and consists of one slab
bearing two spatially distinct series of articulated vertebrae. One of the
series (hereafter referred to as MB.R.1988a) displays 19 articulated dorsal
vertebrae with ribs (12.5 cm in length) associated with a humerus. The other
series (MB.R.1988b) consists of 13 posterior dorsal vertebrae (7 cm in
length) and is also associated with a humerus. Both vertebral series are
segments of the dorsal section of the spine and thus cannot possibly belong
to the same individual. MB.R.1988a is recognized as Stereosternum based on the presence
of 19 dorsals, which exceed the total number of dorsal vertebrae present in
either Brazilosaurus or Mesosaurus (both 18), as well as based on the moderate pachyostosis of its ribs. By
contrast, the identification of MB.R.1988b can only be speculative due to
the absence of informative characteristics, and it is hereafter assumed that it
belongs to Stereosternum like its neighbouring specimen.
Measurements of sampled specimens (in mm) of
Stereosternum and Brazilosaurus (BSPG 1965 I 131).
Abbreviations: fe, femur; hu, humerus; mbl, measured body length.
Specimen number/Body length/% of max bodyHumerusFemurSampled bones% of max body size*calculated/length (690)lengthlengthStereosternumIGPB R 623>65094 % (mbl)3239humerus femur*724 (fe)84 % (hu)dorsal rib105 % (fe)dorsal vertebraeBSPG 1975 I 165>50cm112 % (hu)3635humerus radius*716 (hu and fe)95 % (fe)dorsal ribsMB R 560569 cm100 %3837haemal archSMNK Pal 3808>64cm103 % (hu)3935femur *683 (hu and fe)95 % (fe)dorsal ribMB R 289764 cm92.8 % (mbl)3340dorsal rib86.8 % (hu)108 % (fe)SMNK Pal 3806>63cm89.5 % (hu)3434femur *626 (hu and fe)92 % (fe)dorsal ribsIGPB R 62261088 % (mbl)26.728.1humerus dorsal83.4 % (hu)dorsal rib76 % (fe)dorsal vertebraeMB R 1988a*604 (hu)87.5 % (hu)28NAhumerusdorsal ribSMNK Pal 9165>51cm79 % (hu)3032dorsal ribs*571 (hu and fe)86.5 % (fe)MB R 1988b*517.5 (hu)75 % (hu)24NAhumerusdorsal ribBrazilosaurusBSPG 1965 I 131>26cm68.4 % (hu)2631dorsal ribs*526 (hu and fe)84 % (fe)
Histological features and growth record of Stereosternum and
Brazilosaurus (BSPG 1965 I 131). Abbreviations: cc, calcified cartilage; eb, endosteal
bone; gm, annual growth marks/rest lines; LAGs, lines of arrested growth; lb, lamellar bone; pfb,
parallel-fibred bone; po/pos, primary osteon(s); sl, sharp line separating the
periosteal from the endosteal domain; sec. ost, secondary osteons; svc, simple vascular canals. The percentage
value in the second column refers to the surface ratio that the medullary region
(i.e. usually filled by endosteal bone) occupies when compared to the
cortical region.
Specimen numberMedullary regionBone tissueVascularizationPeriosteal erosion/Growth recordsecondary osteonsStereosternumIGPB R 623young, no distinct LAG, maybe died in its second or third yearHumerus (not midshaft; circular cross section)81.37 %, no sl, high amounts of cc and ebthin cortex highly organized pfbavascularnone, sec. ost.2 annuliFemur∼11.5 %, no sl, eb, no ccinner cortex pfb, outer cortex higher organized pfb and lbavascularnone, sec. ost.1 indistinct rest line, supported by a change in tissue organizationProximal rib sample11.67 %, sl, some remains of cc between ebpfb and lbwell-vascularized inner and middle cortex, svc and posscattered, moderate amount of sec. ost.4 changes in tissue but no distinct rest lineMedian rib sample16.2 %, sl, some cc between ebpfb and lbwell-vascularized inner and middle cortex, svc and posscattered, moderate amount of sec. ost.1 change in tissue and 1 indistinct rest lineBSPG 1975 I 165died in its fourth to fifth year of lifeHumerus (triangular cross section; not exactly sampled at midshaft)24.3 %, sl, no ccinner cortex pfb, outer cortex higher organized pfb and lbmoderate vascularization; svc and pos at the inner cortex of one bone side, more scattered at the postaxial sidefew scattered sec. ost.3 major gm (LAGs accompanied by multiple rest lines) and one change in bone tissue in the inner cortexRadius28.6 %, sl, no cchighly organized pfb/lbavascularnone, sec. ost.multiple rest lines, 4 major cycles are identified and one change in bone tissue in the inner cortexProximal rib (oval)6.7 %, sl, no cchighly organized pfb/lbavascularfew scattered sec. ost.multiple rest lines, 4 major cycles are identified and one change in bone tissue in the inner cortexProximal rib (round)6.5 %, sl, no cchighly organized pfb/lbavascularfew scattered sec. ost.multiple rest lines, 4 major cycles are identified and one change in bone tissue in the inner cortexMB R 5605died in its fourth to fifth year of lifeHaemal arch4 double LAGsProximal3 %, sl, some ccpfbavascularnone, sec. ost.2 indistinct gmDistal40.6 %, sl, no cchighly organized pfb/lbavascularfew erosion cavities sec. ost.3–4 gm
Continued.
Specimen numberMedullary regionBone tissueVascularizationPeriosteal erosion/Growth recordsecondary osteonsSMNK Pal 3808died in its fifth year of lifeHumerus (midshaft; circular cross section)7.2 %, no sl, few remains of ccpfblow vascularization; few scattered svc and posfew scattered sec. ost.3 gm multiple rest linesFemur (not midshaft)68.34 %, sl, high amount of cchighly organized pfbavascularfew scattered sec. ost.1 gmProximal rib sample6.7 %, sl, no cchighly organized pfb/lblow vascularization; few scattered svc and posfew scattered sec. ost.3 major cycles, each consisting of multiple rest lines; third one consists of three widely spaced rest lines in all four samplesMB R 2897died in its fourth year of lifeProximal rib15 %, sl, no cchighly organized pfb/lbmoderately vascularized svc and few posfew erosion cavities; scattered sec. ost.3 LAGs accompanied by tissue changesSMNK Pal 3806died in its sixth year of lifeFemur (not midshaft)43 %, sl, few remains of cchighly organized pfb/lblow vascularization svc and few posfew scattered sec. ost.4 growth mark (multiple rest lines)Proximal rib sample9.15 %, sl, no ccvascularized part loose organized pfb, avascular part highly organized pfb/lbmoderate vascularization (svc and pos) in one direction, avascular at the other sidescattered sec. ost. in one direction5 growth marks (multiple rest lines)Median rib sample11.74 %, sl, high amounts of ccvascularized part loose organized pfb, avascular part highly organized pfb/lbmoderate vascularization (svc and pos)scattered sec. ost.5 growth marks (multiple rest lines)IGPB R 622died in its first year of lifeHumerus (midshaft but slightly compressed; elliptical cross section)20.18 %, sl, locally restricted remains of cccc, pfbavascularsome very large erosion cavities0 gmProximal rib4.1 %, sl, no ccpfbavascularfew erosion cavities grading into sec. ost.0 gmMedian rib25 %, sl, high amounts of ccpfbavascularfew erosion cavities grading into sec. ost.MB R 1988adied in its seventh year of lifeHumerus (midshaft; elliptical cross section)no sl, 14.4 % med. reg., filled by eb, no cchighly organized pfb/lbavascularfew scattered erosion cavities6 major gm (double–multiple rest lines)Proximal rib6.2 %, no sl, no cchighly organized pfb/lbavascularnone6 major gms (multiple rest lines)SMNK Pal 9165died in its fourth year of lifeProximal rib6.96 %, sl, no cchighly organized pfb/lbavascularnone3 LAGs (double ones) plus 1 tissue change in the inner cortexProximal rib4.84 %, sl, no cchighly organized pfb/lbavascularnone3 LAGs (double ones) plus 1 tissue change in the inner cortex
Continued.
Specimen numberMedullary regionBone tissueVascularizationPeriosteal erosion/Growth recordsecondary osteonsMB R 1988bdied in its first or second year of life (or is maybe even younger)Humerus (midshaft; circular cross section)12.6 %, sl, no ccpfbmoderately vascularized radial and longitudinal svcnone4 changes in colour/tissue, 1–2 indistinct rest linesProximal rib12 %, sl, no ccpfbavascularnone4 changes in color/tissue, 1–2 indistinct rest linesBrazilosaurusBSPG 1965 I 131died in its seventh year of lifeProximal rib24 %, sl, no cchighly organized pfb/lbavascularnone6 major gms; multiple, closely spaced rest linesProxi-median rib26 %, sl, locally remains of cclbavascularfew scattered sec. ost.multiple, closely spaced rest linesMedian rib41 %, sl, locally remains of cclbavascularnonemultiple, closely spaced rest linesBody size and ontogenetic stages of specimens
In our sample the maximum body length of a Stereosternum individual is 69 cm (MB.R.5605;
Table 1). Because humerus and femur length are available for this specimen
we use MB.R.5605 as the reference specimen (100 %) for our body length
calculations (Table 1). However, body proportions and ratios seem to be
quite variable among mesosaurs (Table 1), and our reconstructions of body
length in Stereosternum have to be used with care and are best regarded as a rough
estimation. For instance, MB.R.2897, which is overall smaller than
MB.R.5605, has longer humeri and femora than the latter. Besides, several
calculated body lengths based on either humerus or femur dimensions turn out
to be smaller than the actual measured body length (Table 1).
All the sampled individuals are larger than 50 cm and thus well beyond
65 % of the maximum known body length. Based on size, the state of
ossification of carpal and tarsal bones, and the degree of fusion of
cranial and axial elements, most of our specimens are estimated to be
adults, i.e. have reached skeletal maturity. Only SMNK Pal 9165 is not fully grown based on
these morphological characteristics.
Composite microscopic photographs of humeral
cross sections of different individuals of the mesosaur
Stereosternum in normal (a, c, e, g, i) and polarized light (b, d, f, h, j).
(a, b) Humerus cross section of specimen BSPG 1975 I 165. Sample
location is not exactly midshaft. (c, d) Humerus cross section of
specimen SMNK Pal 3808. Sample location is exactly midshaft. (e, f) Humerus cross section of specimen IGPB R 622. Sample location is not exactly
midshaft. (g, h) Humerus cross section of specimen MB.R.1988a.
Sample location is exactly midshaft. (i, j) Humerus cross section
of specimen MB.R.1988b. Sample location is nearly midshaft. Note the sharp
line surrounding the medullary region, which separates the endosteal from
the periosteal domain in humerus BSPG 1975 I 165, IGPB R 622, and MB.R.1988b (indicated by arrows).
ResultsShape of cross sections
The shape of the humeral cross sections is variable, spanning from
triangular (BSPG 1975 I 165) to elliptical (IGPB R 622, MB.R.1988a) and
circular (SMNK Pal 3808; MB.R.1988b) (Fig. 1; Table 2). Although some
dorsoventral compaction of bones has occurred during fossilization, none of
the humeri show a crushed inner bone structure. The morphology of the shaft
affects the apparent histology, and in particular the readability of the growth
record. If this shape is oval or circular, the growth record is regularly
preserved all around the cross section; if it is somehow angled or more
triangular, growth marks tend to merge where the cortex becomes thinner and
to split where the cortex gets thicker.
Composite microscopic photographs of femoral
cross sections of different individuals of the mesosaur
Stereosternum in normal (a, c, e) and polarized light (b, d, f). (a, b) Femur cross section of specimen IGPB R 623. Sample location is not exactly
midshaft. (c, d) Femur cross section of specimen SMNK Pal 3808.
Sample location is distal to midshaft. Note the high amounts of calcified
cartilage (e, f) Femur cross section of specimen SMNK Pal 3806.
Sample location is distal to midshaft. Note the sharp line surrounding the
medullary region, which separates the endosteal from the periosteal domain
in non-midshaft samples of femur SMNK Pal 3806 and SMNK Pal 3808 as well as
in radius BSPG 1975 I 165 (g, h) (indicated by arrows).
Microscopic photographs of cross sections of rib
samples of different individuals of the mesosaur Stereosternum. The variability of shapes
of rib cross sections is due to different sampling location along the
proximal to the median part of the rib shaft as well as due to different
anatomical position along the trunk region (see also Fig. S1).
Proximal to median rib sample of IGPB R 623 in normal (a) and
polarized light (b). Proximal rib sample of MB.R.1988b in normal (c) and polarized light (d). Proximal to median rib sample
of SMNK Pal 3806 in normal (e) and polarized light (f).
Proximal part of haemal arch of MB.R.5606 in normal (g) and
polarized light (h). Distal part of haemal arch of MB.R.5606 in
normal (i) and polarized light (j). Dorsal vertebra in
normal (k) and polarized light (l).
The cross section of the radius of BSPG 1975 I 165 is egg-shaped (Fig. 2g,
h). Femoral cross sections of SMNK Pal 3806 and SMNK Pal 3808 are
sub-circular (Fig. 2c–f), but sampling was not carried out at midshaft.
Only half of the cross section of femur IGPB R 623 (midshaft sample) is
preserved (Fig. 2a, b). Its semicircular shape suggests that the complete
section would have been elliptical. The shape of all rib cross sections is
rather uniform, varying from a pointed oval in the proximal end to round in
the median part (Figs. 3; S1 in the Supplement).
Microanatomy
All sampled long bones share the lack of an inner medullary cavity. Instead
they accommodate a medullary region entirely filled with endosteal bone
(Figs. 1, 2). The farther from midshaft the sample was taken, the larger the
medullary region (Table 2). Bone compactness of midshaft samples of long
bones is 96 % in the well-vascularized humerus MB.R.1988a (Fig. 1i, j)
and in all others 100 %.
Composite microscopic photographs of rib samples
of Brazilosaurus (BSPG 1965 I 131). Proximal part of dorsal rib in normal (a) and
polarized light (b). Proximal to median part of dorsal rib in
normal (c) and polarized light (d). Median part of dorsal
rib in normal (e) and polarized light (f).
Every proximal rib sample (except for that of IGPB R 623, which lacks any
cavity; Fig. 3a, b) bears a very small sized, round medullary cavity (Fig. 3) surrounded by a moderately sized medullary region filled with endosteal
bone (the free medullary cavity together with the surrounding medullary
region make between 4 % and 15 % of the entire cross section, Table 2), while
the enlarged medullary regions (about 25 % of cross section) of medial rib
samples are completely occupied by endosteal bone (e.g. Fig. S1m, n).
The proximal rib sample of Brazilosaurus displays a large medullary region, which
occupies about 24 % of the entire cross section, whereas this region
represents about 41 % of the median sample (Figs. 4, 8a). Bone compactness
of rib samples of Stereosternum is about 97 % in the well-vascularized ribs of MB.R. 2897, SMNK Pal 3806, 98.5 % in proximal rib IGPB R 623, and 100 % in all
others, including the ones of Brazilosaurus.
The proximal part of the haemal arch of MB.R.5605 has only a very
small medullary region (∼3 % of cross section) that
contains one large erosion cavity, whereas the distal part displays a large
medullary region (41 % of cross section) (Fig. 3g–j).
All sampled vertebrae, centra and neural arches alike, depict large compact
endosteal territories filled with small erosion cavities but lack larger
cavities (Fig. 3k, l).
In all bones, compact medullary regions are surrounded by a compact cortex,
resulting in high bone compactness values and clearly indicating
osteosclerosis.
Details of medullary regions in long bones of
Stereosternum in polarized light.
(a) Medullary region depicting clusters of calcified cartilage
between the endosteal bone in midshaft sample of humerus SMNK Pal 3808.
(b) Medullary region depicting endosteal bone and a sharp line
(sl) but no calcified cartilage in humerus BSPG 1975 I 165. (c) Medullary region depicting endosteal bone but no calcified cartilage and
sharp line in midshaft sample of humerus MB.R.1988a. (d) Medullary
region consisting of endosteal bone in midshaft sample of humerus MB.R.1988b. (e) Medullary region consisting of endosteal bone with
clusters of calcified cartilage at the margin of the sharp line in
non-midshaft sample of humerus IGPB R 622. (f) Enlarged medullary
region consisting largely of calcified cartilage and the reduced cortex in
non-midshaft sample of humerus IGPB R 623. (g) Non-midshaft sample
of femur SMNK Pal 3806. The medullary region is enlarged but consists mainly
of endosteal bone, only clusters of calcified cartilage occur. (h) Detail of (g). Abbreviations: cc, calcified cartilage; eb, endosteal bone;
sl, sharp line.
Histological descriptionMedullary regions and occurrence of calcified cartilage in the
endosteal domain
When sampling has not been carried out exactly at midshaft in long bones,
the endosteal and periosteal domains are demarcated by a sharp line (see
Klein and Griebeler, 2018) (Table 2). Except for femur IGPB R 623, humeri
SMNK Pal 3808 and MB.R.1988a, and the proximal rib sample of MB.R.1988a,
all samples show such a sharp line (Table 2), meaning that the best sampling
location is very restricted. The medullary regions of Stereosternum samples consist of
endosteal bone and only a few samples show calcified cartilage (Table 2).
Calcified cartilage occurs in the endosteal domains of the vertebral centra
(Fig. 3k, l) and along their notochordal canal through the centrum. In ribs
and long bones, high amounts of calcified cartilage are found only in
non-midshaft humerus and femur samples (IGPB R 623, Fig. 5f; SMNK Pal 3808),
in the proximomedial rib of IGPB R 622, and in one median rib sample of SMNK Pal 3806. Moderate remains of calcified cartilage occur in the proximal and
median rib of IGPB R 623, whereas the femur of the same individual shows no
calcified cartilage at midshaft. Calcified cartilage occurs locally
restricted in the humeri of IGPB R 622 (Fig. 5e) and SMNK Pal 3808 (Fig. 5a), in the non-midshaft femur sample SMNK Pal 3806 (Fig. 5g), and in the
distal sample of the haemal arch (MB.R.5606, Fig. 3i, j). Bones of
individuals BSPG 1975 I 165 (Fig. 5b), MB.R.1988a (Fig. 5c), and MB.R.1988b
(Fig. 5d) as well as the rib samples MB.R.2897 and SMNK Pal 9165 do not show
any calcified cartilage. Thus, there is no clear pattern (i.e. sampling
location) associated with the presence of calcified cartilage in our sample
of Stereosternum.
The proximal sample of the Brazilosaurus rib does not show any calcified cartilage, but
the medial one bears some locally restricted clusters in its medullary
region (Fig. 4f).
Details of Stereosternum long bone tissue all in polarized light/under
crossed nicols. All showing parallel-fibred bone tissue in
different degrees of organization. (a) Avascular, highly organized
parallel-fibred tissue in femur IGPB R 623. (b) Avascular, highly
organized parallel-fibred tissue partially grading into lamellar tissue in
humerus BSPG 1975 I 165. (c) Some vascularization by incipient
primary osteons in a matrix of parallel-fibred tissue in the inner cortex of
humerus SMNK Pal 3808. (d) Simple vascular canals and incipient
primary osteons in a matrix of parallel-fibred tissue in the outer cortex of
femur SMNK Pal 3806. (e) Simple vascular canals and lightly organized
parallel-fibred tissue in humerus IGPB R 622. (f) Highly
organized avascular parallel-fibred tissue partially grading into lamellar
tissue in humerus MB.R.1988a. (g) Lightly organized parallel-fibred tissue with simple vascular canals in humerus MB.R.1988b.
(h) Highly organized parallel-fibred tissue partially grading into
lamellar tissue in radius BSPG 1975 I 165.
Bone tissue and vascularization in the periosteal domain
In all Stereosternum samples, bone is mainly formed by parallel-fibred tissue. It occurs
in different degrees of organization, from loosely to highly organized (Fig. 6), sometimes partially turning into lamellar tissue (Fig. 6b). Osteocyte
lacunae are large and numerous but often flattened. In long bones and ribs,
vascularization is dominated by simple longitudinally oriented vascular
canals, which are sometimes fully or partially lined with lamellar bone,
resulting in complete or incompletely primary osteons (Klein, 2010; Table 2). Primary osteons are often secondarily widened (Figs. 6d, 8b). In some
samples, vascular canals and primary osteons are intermixed with erosion
cavities and/or young (i.e. wide open; Currey, 2002) secondary osteons
(Table 2; Fig. 6d). The humerus of IGPB R 622 bears very large,
irregularly shaped, erosion cavities along its dorso-preaxial and
ventro-postaxial edges (Fig. 6e). The humerus of MB.R.1988b shows the
highest vascular density. Its cortex consists of mainly longitudinally and
some radially arranged simple canals (Fig. 6g). However, most samples are
lightly vascularized or even avascular (Fig. 6a, b, f, h; Table 2). Vascular
density in the ribs is well correlated with that in the long bones in
individual MB.R.1988a and IGPB R 622 (both avascular), SMNK Pal 3808 (low
vascular density), and SMNK Pal 3806 (moderate vascular density) (Figs. 1, 3;
S1). There is no such correlation in BSPG 1975 I 165 and
MB.R.1988b, which both have a well-vascularized humerus but lightly vascularized
or even avascular ribs, or in IGPB R 623, in which on the contrary the femur
is avascular but the ribs are well vascularized. The haemal arch of
Stereosternum is avascular (Fig. 3g–j). Scattered erosion cavities and/or secondary
osteons are the only form of periosteal remodelling, and it does not affect
the completeness of the growth record. Periosteal (dorsal and ventral part)
and endochondral (anterior and posterior part) territories of vertebrae
(sensu de Buffrénil et al., 2008) are distinguishable, but not as
clearly as in eosauropterygians (Klein et al., 2019). The endosteal bone is
more compact than the periosteal bone (Fig. 3k, l). The layer of compact
periosteal cortex is thicker in the ventral part of the centrum than in the
dorsal one. The ventral part of the centrum is traversed by large, simple,
radially running vascular canals, whereas those innervating the neural arch
are rarer, smaller, more irregularly shaped and randomly scattered. The
dorsal vertebrae clearly show a notochordal canal through the centrum,
occupied by cartilage cells.
The rib samples of Brazilosaurus display avascular lamellar bone (Table 2; Fig. 4).
Growth mark count in humerus MB.R.1988a. Note the
presence of multiple rest lines at the end of cycles as well as the split
and merging of some rest lines.
Growth record
Neither growth marks nor any evidence of cyclical growth is visible in the
vertebrae. Because of the lack of intensive periosteal remodelling in
midshaft samples of long bones and proximal samples of ribs, the growth
record is complete in these bones; i.e. no inner growth marks seem to be
lost due to remodelling. In most specimens of Stereosternum and in the ribs of
Brazilosaurus, annual growth cycles are represented by distinct lines of arrested growth
(LAGs). These are often accompanied by additional rest lines (Fig. 7),
resulting in the presence of double or multiple lines at the end of each
annual growth cycles, making an exact count difficult. Furthermore, a single
LAG can split and further merge back with itself again (Fig. 7). This, along
with the absence of changes in tissue organization or vascularity, hampers
annual growth mark count. The growth mark count for each sample is given in
Table 2. In general, the number of growth marks seems to correspond in long
bones and ribs of the same individual (Table 2).
Yet, in two humeri of Stereosternum individuals (IGPB R 623, Fig. 1e, f; MB.R.1988b, Fig. 1i, j) and one rib sample (IGPB R 623, Figs. 3a, b, S1m, n),
growth marks are not represented by distinct LAGs. Instead, the cortex is
stratified by accumulations of osteocyte lacunae to thin layers and/or by a
change in bone tissue organization. Because all other samples of
Stereosternum show distinct LAGs, these less pronounced growth marks, mainly those in
MB.R.1988b, are not counted as annual growth marks but interpreted as
subcycles stratifying the first year of life.
In bones of individual BSPG 1975 I 165 (Figs. 1c, d, 2g, h, S1a,
b) and in the ribs of SMNK Pal 9165 (Fig. S1i, j), the innermost
cortex also contains a large inner first cycle, which contains a change in
tissue that is then followed by clear LAGs accompanied by multiple rest
lines. This inner change in tissue is also not counted as an annual growth mark
but interpreted as a sub-cycle, dividing the fast and large growth period in
the first year of life. This large inner first cycles indicate that most
(up to 50 %) of the appositional growth happened in the first year of life in
Stereosternum.
Individual SMNK Pal 3808 shows three major growth cycles, each consisting of
multiple rest lines (Fig. 1c, d). The third of these consists in the humerus
and in the rib sample (Fig. S1g, h) of three widely spaced rest lines.
Neither an increase in tissue organization, a decrease in vascular density,
nor a change in the spacing of rest lines is identified in any sample, and
thus nothing can be said about onset of sexual maturity. Because in most
samples any annual cycle ends in multiple rest lines, resembling a kind of
external fundamental system, addressing the attainment of full growth is also
not possible with certainty in our sample. However, only bones of individual
SMNK Pal 3806 show multiple rest lines in their outer cortex, meaning that
it died at the end of a growth cycle, whereas all others died sometime
during the growth cycle.
The rib samples of Brazilosaurus depict a high number of closely spaced rest lines
throughout the entire cortex, making it difficult to identify annual cycles.
DiscussionComparison of mesosaur histology and microanatomyGeneral comparison
Bone tissue in Stereosternum and Brazilosaurus is lamellar-zonal, with a low (in some rare cases,
moderate) degree of vascularization or being avascular. This highly organized and lightly vascularized type of bone tissue is for example characteristic of modern
reptiles, of Claudiosaurus (de Buffrénil and Mazin, 1989) and small choristoderes
(Skutschas and Vitenko, 2017). However, sauropterygians from the Triassic
present less organized and more vascularized bony tissues, suggesting higher
growth rates than mesosaurs (Klein, 2010; Hugi et al., 2011; Klein and
Griebeler, 2018). The high degree of bone compactness in sampled long bones
of Stereosternum is unique among marine reptiles. It exceeds that of the Permian
Claudiosaurus (de Buffrénil and Mazin, 1989), of most Triassic marine reptiles (Klein
et al., 2015a, 2016; Klein and Griebeler, 2018), and of the modern marine
iguana Amblyrhynchus (Hugi and Sánchez-Villagra,
2012). Only pachypleurosaurs from the Alpine Triassic have comparable bone
compactness values, although these are on average somewhat lower than that
of Stereosternum (Hugi et al., 2011).
The large number of osteocyte lacunae in vertebrae samples of
Stereosternum following the direction of bone growth in the endochondral bone is not
observed in marine reptiles (Klein et al., 2019; Tanja Wintrich, personal observation) and is
thus very distinctive, too.
Comparison of the microanatomy of mesosaur ribs
Although sample size is restricted for Brazilosaurus and Mesosaurus, and as we did not study the
ribs of Mesosaurus firsthand, we want to mention here some preliminary observations,
resulting from comparing ribs of mesosaurs. Information about the ribs of
Mesosaurus is drawn from Nopcsa and Heidsieck (1934) and de Ricqlès (1974).
Mesosaurus, Stereosternum, and Brazilosaurus share highly osteosclerotic ribs as a result of condensation of the
periosteal cortex and the medullary region, with both domains lacking any
spongiosa. However, as the current study documents, the surface ratio of
periosteal cortex over medullary region is broader in Stereosternum (≥85 %–95 % in
proximal rib samples/75 % in median rib samples) than in Brazilosaurus (≥75 % in
proximal rib samples/60 % in median rib samples) (Table 2; Fig. 8).
According to Nopcsa and Heidsieck (1934, p. 442) as well as to de Ricqlès
(1974, fig. 11), the medullary region in the ribs of Mesosaurus is nearly as broad as
the periosteal cortex (Fig. 8c–e; ∼64 % measured from de
Ricqlès, 1974, fig. 11). It is unclear whether these ribs of Mesosaurus were sampled
proximally or medially, but in terms of the ratio between the endosteal and
periosteal domain, the ribs of Mesosaurus clearly differ from those of Stereosternum and are in
this regard rather similar to those of Brazilosaurus (Fig. 8).
Comparison of mesosaur ribs. (a)Brazilosaurus (BSPG 1965 I 131) rib samples. (b)Stereosternum (SMNK Pal 3806) rib samples. (c)Mesosaurus rib sample and outline sketch modified from de Ricqlès (1974).
(d)Mesosaurus rib sample modified from Nopcsa and Heidsieck (1934).
Brazilosaurus is characterized by a highly organized avascular lamellar tissue, regularly
stratified by very clear rest lines. This tissue type is easily
distinguished from that of Mesosaurus and Stereosternum (Figs. 3, 4, 8). This characteristic
stratification in Brazilosaurus seems to be independent of ontogenetic age. As for them,
Mesosaurus and Stereosternum share comparable tissue types in ribs. They both present a less
organized tissue and some vascularization in the form of longitudinally simple
vascular canals and primary osteons. Comparatively to Brazilosaurus, they are less
regularly stratified by rest lines.
Thus, considering the relative size of periosteal and endosteal domains as
well as the tissue type, and provided sampling was carried out in similar
locations, the ribs of each taxon are very characteristic of and distinct from
each other.
Pachyostosis and osteosclerosis in mesosaurs
Nopcsa and Heidsieck (1934) already pointed out that Stereosternum ribs are less
pachyostotic and osteosclerotic than Mesosaurus. In terms of pachyostosis in the ribs,
our specimens confirm that this genus stands somewhat in between the two
other mesosaur genera, with its ribs being less pachyostotic than those of
Mesosaurus but more so than those of Brazilosaurus. We show that, albeit gracile, the ribs of
Brazilosaurus are also osteosclerotic. Thus, contrary to pachyostosis sensu stricto (Houssaye, 2009),
osteosclerosis affects the ribs of all three species to similar degrees. We
further document osteosclerosis for the first time in the long bones of
Stereosternum. The presence of osteosclerosis in all our sampled bones without pronounced
pachyostosis allows us to conclude that osteosclerosis occurs early on in
ontogeny.
As mentioned above, Nopcsa and Heidsieck (1934) had described pachyostosis
and osteosclerosis in the ribs of Stereosternum and Mesosaurus. They mentioned an increase in
the periosteal cortex and a highly condensed medullary region for both taxa.
According to these authors, the medullary region was condensed by endosteal
deposits in the ribs of Mesosaurus. In their Stereosternum sample, however, the centre of the rib
contained high amounts of calcified cartilage. Because the ribs were also
small in size, they argued that their Stereosternum specimen was likely a young
individual. They were convinced that the microanatomy in older ontogenetic
stages of Stereosternum would look like that in their sample of Mesosaurus (Nopcsa and Heidsieck,
1934, p. 444). This is confirmed by our study, where individuals larger than
65 % of the average adult length do not show high amounts of calcified
cartilage. Besides, in our Stereosternum sample the presence or absence of calcified
cartilage is not related to sampling location or specimen age (Table 2). Some individuals retain it, whereas others do not. Our results confirm the
observations of Nopcsa and Heidsieck (1934) that endosteal deposits in the
medullary region of ribs are the main driver of osteosclerosis, as is the
case in all other bones of Stereosternum.
Contrary to Nopcsa and Heidsieck (1934), de Ricqlès (1974) and de
Ricqlès and de Buffrénil (2001) suggested that osteosclerosis in the
ribs of Mesosaurus might have been caused by an incomplete endochondral ossification
and the retaining of calcified cartilage. Our results do not support this
hypothesis, but it is possible that the observations of de Ricqlès
(1974) were nonetheless accurate if the specimen of Mesosaurus he studied was at an
early ontogenetic stage, or if the presence of calcified cartilage in bones
of Mesosaurus is as variable as in Stereosternum.
The absence of high amounts of calcified cartilage in Stereosternum and Brazilosaurus is comparable to
what is known for other aquatic or semi-aquatic reptiles. In the Permian
diapsid Claudiosaurus, osteosclerosis is also the result of intensive endosteal deposits
and Haversian reconstruction (de Buffrénil and Mazin, 1989), whereas
endochondral ossification is quite complete (de Ricqlès and de
Buffrénil, 2001). In the case of small Early Cretaceous choristoderes,
Skutschas and Vitenko (2017) showed that osteosclerosis was achieved through
endosteal deposits as well, but because the humerus they studied was likely
not sampled at midshaft, this result in choristoderes might not be
comparable to ours on mesosaurs. Triassic pachypleurosaurs, contrary to the
three previously mentioned clades, retain high amounts of calcified
cartilage at the midshaft of long bones and in their ribs (Klein et al., 2019).
All the aforementioned taxa are of comparably small size and are
interpreted as having inhabited shallow marine or lacustrine environments.
Despite this relative homogeneity in their life conditions, osteosclerosis
in these clades is achieved in different ways. Thus, there does not seem to
be any correlation between environmental or size-related factors and modes
of osteosclerosis. Whether these processes are influenced by phylogeny or
correlate with other factors must be clarified in future studies.
According to the few phylogenetic studies regarding the intra-relationships
of Mesosauridae, Brazilosaurus represents the basalmost taxon of the clade (Rossmann and
Maisch, 1999; Karl et al., 2007). While this taxon is also the least
pachyostotic, it nevertheless displays an advanced degree of osteosclerosis.
Thus, pachyostosis and osteosclerosis did not occur simultaneously in the
evolutionary history of the clade but rather as two distinct steps. The
fact that osteosclerosis occurred first suggests that increasing inner bone
compactness was more crucial and possibly easier to achieve than increasing
bone volume early on in the adaptation to an aquatic lifestyle. In this
regard, Brazilosaurus is less adapted to an aquatic environment, and thus less derived,
than Stereosternum and Mesosaurus.
Life history traits
Thanks to the lack of periosteal remodelling, the growth record in
Stereosternum specimens is complete. The first growth cycle is always the largest, whereas
most of the other cycles are closer and relatively evenly spaced.
Considering that, based on the length of its humerus, the newborn
Stereosternum described by Bickelmann and Tsuji (2018) would have had a body length of
about 13–14 cm; and that some of our adult individuals measure between 60 and 70 cm (Table 2); and given the disparity in growth cycle width in their bones,
it is safe to assume that Stereosternum would have experienced an enormous increase in
size during its first years of life. This is comparable to juveniles of
extant lepidosaurs, who can double their size in their first year (Andrews,
1982; Lin and Rieppel, 1998), or with Alligator mississippiensis, whose apposition rates peak during
its first year of growth as well (Woodward et al., 2014). However, some
specimens of Stereosternum reached a body length of more than 60 cm in their first or
second year of life (Table 2) – an extreme leap in body size in a short
time, especially considering the highly organized and lightly vascularized
tissue – whereas other individuals needed up to 4 or more years to reach the
same size.
As a result of this observation, Stereosternum shows a poor correlation between growth
mark count (i.e. age) and body length (Tables 1, 2). One of the largest
individuals (IGPB R 623, 94 % of the maximum body size) in the sample exhibits
only two annuli that are not very distinct (Figs. 2a, b; 6a). Another
relatively large individual (IGPB R 622, 88 % of the maximum body size) does
not show any growth marks (Figs. 1e, f; 6e), whereas an individual at
87.5 % of the maximum body size (MB.R.1988a; Figs. 1g, h; 6f; 7) has six
major growth cycles. Ribs of SMNK 9165 already show three growth marks, but
on the basis of morphology it is not yet fully grown. This poor correlation
between age and size might be related to several factors such as high
intraspecific variation, developmental plasticity, or sexual dimorphism.
However, biogeographic and/or time-related differences might be also an
explanation for this discrepancy because as a result of time-averaging our
specimens were likely not members of the same populations. They originated
from different localities in the widespread Irati Formation, and
stratigraphic as well as environmental and climate differences cannot be
excluded. It is also possible that growth marks reflect a non-yearly
seasonality that would vary much more drastically depending on the
environment, hence biasing the age estimate.
No indication of onset of sexual maturity was detected in the here-studied
mesosaur individuals, which in other tetrapods is often indicated by a
change in tissue and/or vascular organization (i.e. change in growth rate)
or by a different spacing pattern of LAGs (i.e. cycle distance) (e.g. Klein
and Griebeler, 2018).
Whether any of our individuals had achieved maximum size and thus stopped
growing could also not be determined. The outer cortex of most samples shows
avascular, highly organized parallel-fibred bone or lamellar bone. Annual
growth cycles always end with multiple rest lines, which is thus not a good
criterion for full growth in the current sample.
The presence of multiple rest lines at the end of each cycle indicates
strong influence of exogenous and endogenous factors. It thus could
represent two or several growth seasons (Castanet et al., 1993) or two or
more reproduction cycles per year. The occurrence of multiple rest lines in
this extent has previously been described, for example, for Triassic temnospondyls
(Konietzko-Meier and Klein, 2013) and sauropterygian placodonts (Klein et
al., 2015b). It is also described for modern newts (Francillon-Vieillot et
al., 1990) and sea turtles (Snover and Hohn, 2004).
Based on growth mark count, the oldest of the studied Stereosternum individuals died in
its seventh year.
The growth mark count on the ribs of the Brazilosaurus specimen indicates it had reached
a similar age.
Conclusions
All bones of Stereosternum show strong osteosclerosis, and so do, somewhat unexpectedly,
the gracile ribs of Brazilosaurus. In Stereosternum and, as far as we can evaluate on the basis of a
two-rib sample, in Brazilosaurus, osteosclerosis is reached by intensive endosteal
deposits, usually coupled with fairly complete erosion of calcified
cartilage, and the inhibition of periosteal remodelling. Comparison of our
results of rib histology and microanatomy with similar published data on
Mesosaurus (Nopcsa and Heidsieck, 1934; de Ricqlès, 1974) reveals that the ribs of
Mesosaurus, Stereosternum, and Brazilosaurus can be well distinguished histologically.
Our results further confirm an aquatic adaptation (i.e. strong
osteosclerosis) for individuals of Stereosternum and Brazilosaurus over 65 % body length, favouring
an aquatic lifestyle. Canoville and Laurin (2010) suggested an aquatic lifestyle for Mesosaurus as well, whereas Núñez Demarco et al. (2018) concluded an
aquatic lifestyle only for juveniles and young adults of Mesosaurus but postulated a
terrestrial lifestyle for more mature individuals based on vertebrae
morphology and morphometric data. In any case, the lifestyle of
Stereosternum might have differed from that of Mesosaurus, because Mesosaurus is the only mesosaur that has
pachyosteosclerotic bones.
The study of the growth record of Stereosternum reveals a poor correlation of body size
and age in years. Some individuals of Stereosternum achieved most of their body size
within their first or second year of life, whereas others needed several years.
These observations may point to strong developmental plasticity. Life
history traits such as the onset of sexual maturity or attainment of full size
are not identifiable in the growth record. The maximal growth mark count in
our sample is 6, suggesting that these individuals died in their seventh
year of life.
Striking is the presence of double or even multiple rest lines at the end of
each cycle, making exact growth mark counts difficult and pointing to a high
influence of exogenous (e.g. several growth season per annum) and endogenous
(e.g. several reproduction cycles per annum) factors.
In conclusion, our results provide novel insights and reveal differences in
the process of aquatic adaptation in mesosaur taxa. Individuals of
Stereosternum, having reached more than half of the maximum known body length, do not yet
show pachyostosis of their ribs or other bones but already display distinct
osteosclerosis. It is very interesting to note that osteosclerosis also
occurs in Brazilosaurus, the most basal member of Mesosauridae, lacking proper
pachyostosis and thus being less adapted to an aquatic environment when
compared to Stereosternum and Mesosaurus.
Data availability
All thin sections are stored under the respective specimen number in the respective public collection as listed in Table 2. See the section “Institutional abbreviation” for details on repositories.
The supplement related to this article is available online at: https://doi.org/10.5194/fr-22-91-2019-supplement.
Author contributions
NK did the histological and microanatomical
study of long bones and ribs, wrote parts of the manuscript, and prepared
figures. AV did the taxonomical assignment of all specimens except for those
from the GPI, wrote parts of the manuscript, and prepared figures. HS
contributed to the taxonomical assignment of specimens from the GPI
collection and to the introduction. TW did the histological study of the
vertebrae. JF wrote parts of the manuscript. All authors contributed to the
discussion.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
We thank Oliver Rauhut (BSPG), Martin Sander (IGPB), Daniela Schwarz (MB), and Eberhard Frey
(SMNK) for giving us the permission to sample specimens under their care. Olaf Dülfer (IGPB) is acknowledged for the production of thin sections. We are grateful to the helpful comments of the
reviewers Dorota Konietzko-Meier and Sean Modesto, as well as the
editor Florian Witzmann.
Financial support
Part
of this research was funded by a grant from the German Research Foundation
(grant no. DFG FR 2457/6-1) to Jörg Fröbisch.
Review statement
This paper was edited by Florian Witzmann and reviewed by Dorota Konietzko-Meier and Sean Modesto.
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