The earliest undisputed crown-group amniotes date back to the Late Carboniferous, but the fossil record of amniotic eggs and embryos is very sparse, with the oldest described examples being from the Triassic. Here, we report exceptional, well-preserved amniotic mesosaur embryos from the Early Permian of Uruguay and Brazil. These embryos provide the earliest direct evidence of reproductive biology in Paleozoic amniotes. The absence of a recognisable eggshell and the occurrence of a partially articulated, but well-preserved embryo within an adult individual suggest that mesosaurs were viviparous or that they laid eggs in advanced stages of development. Our finds represent the only known documentation of amniotic embryos in the Paleozoic and the earliest known case of viviparity, thus extending the record of these reproductive strategies by 90 and 60 Ma, respectively.

Mesosauridae comprises the oldest known aquatic amniotes which lived in Gondwana during the Early Permian. Previous work in the Uruguayan mesosaur-bearing Mangrullo Formation suggested that mesosaurids lived in an inland water body, inferred as moderately hypersaline, with exceptional preservational conditions that justified describing these strata as a Fossil-Lagerstätte. Exquisitely preserved articulated mesosaur skeletons, including gastric content and associated coprolites, from the Brazilian Iratí Formation in the State of Goiás (central-western Brazil) indicate excellent conditions of preservation, extending the Konservat-Lagerstätte designation to both units in the Paraná Basin. The near-absence of more resistant fossil remains, like actinopterygian and temnospondyl bones, demonstrates the faunistic poverty of the mesosaur-bearing “salty sea”. Our studies of the alimentary habits of mesosaurids through the use of stereoscopic microscopy, light and electronic microscopy, and X-ray diffractometry suggest that the diet of mesosaurids was predominantly composed of pygocephalomorph crustaceans (possibly not exceeding 20 mm in length). However, the presence of bones and bone fragments of small mesosaurs in the gastric content, cololites, coprolites, and possible regurgitalites may also indicate cannibalistic and/or scavenging habits. Cannibalism is relatively common among vertebrates, particularly during conditions of environmental stress, like food shortage. Likewise, the apparent abundance of pygocephalomorph crustacean fossils in the Iratí and Mangrullo Formations, outside and within the studied gastric, cololite, and coprolite contents, might have to do with environmental stress possibly caused by volcanic activity, in particular ash spread into the basin during the Early Permian. In this context, casual necrophagy on the dead bodies of small mesosaurs and large pygocephalomorphs might have been an alternative alimentary behavior adopted for survival in mesosaurs.

Mesosaurs have been considered strictly aquatic animals. Their adaptations to the aquatic environment are well known and include putative viviparity, along with the presence of several skeletal characters such as a long, laterally compressed tail, long limbs, the foot larger than the manus, and presence of pachyosteosclerotic bones. They were also described as possessing non-coossified girdle bones and incompletely ossified epiphyses, although there could be an early fusion of the front girdle bones to form the scapulocoracoid in some specimens. Some of these features, however, are shared by most basal tetrapods that are considered semiaquatic and even some terrestrial ones. The study of vertebral columns and limbs provides essential clues about the locomotor system and the lifestyle of early amniotes. In this study, we have found that the variation of the vertebral centrum length along the axial skeleton of Mesosaurus tenuidens fits better with a semi-aquatic morphometric pattern, as shown by comparisons with other extinct and extant taxa. The present study allows us to suggest that whereas well-preserved mesosaur skeletons are mostly represented by juveniles and young adults that inhabited aquatic environments, more mature individuals might hypothetically have spent time on land. This is also supported, to an extent, by taphonomic factors such as the scarce representation and poor preservation of remains of mature individuals in the fossiliferous levels, and also by anatomy of the appendicular bones, and particularly the strongly ossified epiphyses and tarsus.

The hypotheses about the origin of the primitive amniotic tarsus are very speculative. Early studies argued that the origin of the astragalus, one of the largest proximal bones in the tarsus of basal amniotes, was produced by either the fusion of two, three, or even four of the original tarsal bones, the intermedium, the tibiale and the proximal centralia (c4 and c3), or that the intermedium alone transforms into the primitive astragalus. More recent studies have shown that the structure of the tarsus in Captorhinus supports the former hypothesis about a fusion of the intermedium, the tibiale, the proximal centrale (c4) and eventually c3, producing a purportedly multipartite structure of the amniotic astragalus, but the issue remained contentious. Very well preserved tarsi of the Early Permian aquatic amniote Mesosaurus tenuidens Gervais, 1864–1865, which represent the most complete ontogenetic succession known for a basal amniote (the other exceptional one is provided by the Late Permian diapsid Hovasaurus boulei Piveteau, 1926), suggest that there is more than one ossification center for the astragalus and that these fuse during late embryonic stages or maybe early after birth. A non-hatched Mesosaurus in an advanced stage of development shows that the tarsus is represented by a single bone, most probably the astragalus, which seems to be formed by the suturing of three bones, here interpreted as being the intermedium, the tibiale, probably already integrated to the c4 in an earlier stage of the development, and the c3. An amniote-like tarsal structure is observed in very basal Carboniferous and Permian tetrapods such as Proterogyrinus, Gephyrostegus, the diadectids Diadectes and Orobates, some microsaurs like Tuditanus and Pantylus and possibly Westlothiana, taxa that were all considered as true amniotes in their original descriptions. Therefore, the structure of the amniotic tarsus, including the configuration of the proximal series formed by the astragalus and the calcaneum, typically a pair of enlarged bones, could have been established well before the first recognized amniote walked on Earth. Accordingly, the tarsus of these taxa does not constitute specialized convergences that appeared in unrelated groups, they might be instead, part of a transformation series that involves taxa closely related to the early amniotes as some hypotheses have suggested.