When first described, it was hypothesized that the circular traces preserved in basal birds from the Jehol Biota represented remnants of the PFM of mature or nearly mature follicles within the left ovary9. The absence of calcified eggshell in the oviduct suggested that ovulation had not begun in any specimen. In one enantiornithine (STM29-8), the unusual surface texture in the purported follicles was interpreted as the imprints of a well-developed network of blood vessels within a highly vascularized PFM9 (Supplementary Fig. 3).
The results of our analyses support identification of the remains preserved in enantiornithine STM10–12 as remnants of the ovary and a vascularized PFM. The tissues in STM10–12 present the same morphological and histochemical characteristics as those of an avian chordae, a contractile structure made of intertwined collagen fibers with smooth muscle fibers that expel the oocyte during ovulation in extant birds (Fig. 2). STM10–12 also preserves structures morphologically consistent with extant blood vessels (Fig. 3). Given that the analyzed fragments of purported follicles in STM10–12 present virtually all the tissue characteristics (i.e., appropriate size, morphology, and histochemistry) of the three main components found in extant PFMs (i.e., smooth muscle fibers, collagen fibers, and blood vessels) the most parsimonious and plausible conclusion is that the circular structures in STM10–12 are indeed the fossilized remnants of pre-ovulatory ovarian follicles, also consistent with their preserved anatomical location. Fossilized structures morphologically consistent with collagen fibers and muscle fibers have been previously identified in numerous other Mesozoic specimens (e.g., refs. 26,27,28,29,30,31). This study contributes to the mounting evidence that such tissue components can preserve in deep-time.
The fossilized follicles in STM10–12 are by no means completely preserved, but rather represent fragments of these structures. We found no histological evidence of other tissues that are found in the PFM of extant pre-ovulatory follicles, such as non-collagenous fibers of the inner perivitelline membrane, granulosa cells, nerve fibers32, and the ovarian surface epithelium (Supplementary Fig. 1).
Fossilized blood vessels have been previously reported in some specimens of Mesozoic dinosaurs and Cenozoic turtles (e.g., refs. 33,34,35,36). The vessels in STM10–12 are not consistent in morphology with fungal hyphae in that they lack septae and fungal hyphae are much smaller (see refs. 33,34). Given their size and morphology, the most logical interpretation is that these structures are remnants of original blood vessels belonging to an originally highly vascularized PFM.
The fossilization of blood vessels is apparently more common than generally recognized in material from the Mesozoic and these structures have already been thoroughly documented both morphologically and chemically (e.g., refs. 26,29,33,34,37,38,39). In previous studies, fossil blood vessels were mostly observed in three dimensions (3D), photographed as ‘floating’ material in demineralizing solutions, whereas in this study the blood vessels were observed in 2D sections exposing the blood vessels through longitudinal cuts (Fig. 3). In blood vessels analyzed in 3D, branching is also commonly observed (e.g., see refs. 33,38,40) but in contrast, in sectioned vessels branching is much less common (e.g., see Fig. 3f–h), which may explain why very few branching patterns were observed in our sample (only in Fig. 3c).
Noteworthy, blood vessels in STM10–12 were only visible in the demineralized paraffin slides (Fig. 3) but not in the ground-sections nor the SEM images (Fig. 2). A possible logical explanation is that this is due to differences in tissue compaction and distortion between the samples in the ground-sections and the paraffin slides. In the ground sections, the tissues were embedded in resin without being demineralized. Resin-embedded tissues are tightly compacted (Fig. 2d) and do not undergo any significant distortion during preparation. On the other hand, demineralized tissues that get embedded in paraffin go through multiple distortion and tearing events (i.e., during demineralization, processing through different solutions, but especially after being cut on a microtome and placed on top of warm water in the water bath prior to mounting on glass slides, see Supplementary Methods). These distortions create an artificial ‘decompaction’ of the fossil tissues in the paraffin slides (which occurs commonly even while making slides of extant tissues), and this is most likely what enabled the visualization of blood vessels in STM10–12 (Figs. 2g–j and 3) that were not directly visible in ground-sections nor through SEM (Fig. 2d). These results suggest that the three methods employed here (ground-sectioning, SEM, and paraffin histology) yield complementary information and, when used together, can help to provide more rigorous identifications and clarify our understanding of fossilized soft-tissues.
EDS showed that the fossilized soft-tissues preserved in STM10–12 underwent alumino-silicification (Fig. 4). This same process has been reported in fossilized branchiopod (clam shrimp) eggs also from the Jehol Biota, where all the envelopes were made of calcium phosphate but some of the eggs had their internal contents replaced by alumino-silicates41. An explanation for the mechanism of alumino-silicate replacement was not provided for the Jehol clam shrimp eggs41, but we suggest that clay minerals from sediments were involved. Clay minerals have been determined to be important agents in the fossilization of soft-tissues in other settings (such as the Ordovician Soom Shale of South Africa and the Cambrian Burgess Shale of Canada)42,43. In these cases, the soft-tissues were replaced rapidly after death by authigenic clay minerals (via direct precipitation of authigenic clays onto the tissues). It is the most logical explanation for the process of alumino-silicification seen in the tissues of STM10–12, and this process may have happened rapidly after death before extensive tissue decay.
EDS also revealed an enrichment in iron, indicating that the soft-tissues may also have experienced some limited mineralization with iron oxides (Fig. 4, see the low iron levels). The mineralization of soft-tissues via iron oxides (such as goethite and biogenic iron oxyhydroxide) has been reported in Mesozoic dinosaurs38 and a similar process may have occurred as well in STM10–12. A potential source for this iron may be the pyroclastic flows that intermittently interrupted the deposition of lacustrine sediments, and/or microbial mats, as proposed for Jehol invertebrates44,45. Moreover, based on our new data, we also suggest that another source of this iron may be the hemoglobin (a protein found in red blood cells) coming directly from the blood vessels of the organism itself. An experimental study on ostrich blood vessels demonstrated that iron and oxygen from hemoglobin play a key role in tissue stability and in the exceptional preservation of soft-tissues in deep-time38. This hypothesis could be tested in the future with immunohistochemistry and antibodies raised against avian hemoglobin.
Although pyritization (via iron sulfides) has been demonstrated in the soft-body parts of Jehol insects and hypothesized to play an important role in the preservation of soft-tissues in Jehol fossils44,45, the EDS data here do not show any sulfur in the sample, meaning STM10–12 underwent a different fossilization process and that pyritization is simply one of the many processes involved in soft-tissue preservation in the Jehol paleolakes.
All of the histological and histochemical data collected here (Figs. 1–4) demonstrate the exceptional preservation of the soft-tissues in STM10–12 (i.e., fossilized chordae and blood vessels from the PFM), but more precise and more specific chemical analyses (such as synchrotron-FTIR, or immunohistochemistry) are needed to fully characterize the preservation of these tissues at a deeper molecular level.
Until this study, interpretations of the purported follicles as ingested seeds (e.g., see Supplementary Fig. 4) indeed represented a viable alternative hypothesis13,14. Testing these competing hypotheses was imperative to our understanding of both the evolution of the paravian reproductive system (i.e., to confirm whether or not early birds indeed had only one functional ovary like extant birds and lacked strong follicular hierarchy) and digestive system (as yet there is no direct evidence regarding the diet of enantiornithines in the Jehol46,47).
Many morphological arguments have previously been raised against the interpretation of these remains as ingested seeds5. For example, the preserved structures also lack the surficial ornamentation observed in most fossilized seeds (Supplementary Fig. 4). Furthermore, the tissues here identified in STM10–12 (smooth muscle fibers, collagen fibers, and blood vessels; Figs. 2 and 3) are strictly animal tissues and are non-existent in plants. None of the histological slides of STM10–12 reveal tissues reminiscent of fossilized plant material with their characteristic cell walls22,48, from either gymnosperms or angiosperm seed or fruit tissues (e.g., cuticle, seed coat or testa, internal integument layers, and embryonic tissues; e.g., refs. 22,49,50). Although our original intent was to directly compare the tissues in STM10–12 with a fossil seed preserved in the stomach of the holotype of Jeholornis prima (Supplementary Fig. 4), close examination showed that the seeds in this specimen are only impressions, and thus cannot be used for proper comparison with seed tissues. Lastly, the STM10–12 samples were also checked for the presence of phytoliths, which are microscopic structures made of silica that are found in plant tissues and can persist for millions of years after the decay of the plant51, but none were found.
Although the preservation of ovarian follicles in STM10–12 is confirmed, this hypothesis needs to be independently tested in each individual specimen (Supplementary Table 1); as yet, it is still possible some of the purported follicles in other specimens may represent ingested seeds. Confirmation of follicle preservation in one specimen does nonetheless put an end to the controversy regarding whether or not such remains can preserve and confirms that only a single functional ovary was present in at least some non-neornithine avians8,9. Additionally, our results support paleobiological hypotheses based upon the original identification, such as observations regarding the relative sizes of these ovarian follicles. Compared to modern birds only subtle size variations are observed in the fossilized follicles of Jehol birds (meaning follicular hierarchy was absent), leading to the inference that in basal birds yolk deposition occurred much more slowly than in extant bird due to the lower metabolic rates of non-ornithuromorph birds8,9. This study reveals that this was true at least for enantiornithine STM10–12, which suggests this may be similarly true about other non-ornithuromorph birds in which slow growth rates are observed through osteohistology. It is likely that a strong follicular hierarchy is a derived feature of a subset of the Ornithuromorpha (because some of them still retain plesiomorphically slower growth rates), but only direct evidence with preserved follicles in Cretaceous ornithuromorphs could confirm this hypothesis.
The Early Cretaceous Jehol Biota of China preserves one of most extraordinary extinct fauna and flora ever discovered, revealed through a taphonomic environment that was extremely conducive to the fossilization of both hard and soft-tissues1,4. Few histological analyses on preserved soft-tissues exist due to the destructive nature of most of these methods. However, our results demonstrate that these types of analyses can eliminate doubt and help to further understand preservation. Previous taphonomic studies conducted on Jehol invertebrates showed that alumino-silicification and pyritization help preserve soft-tissues in the Jehol41,45. In STM10–12, no pyritization was found, but the tissues apparently underwent alumino-silicification and a slight mineralization with iron, potentially iron oxides. This suggests that soft-tissue fossilization in the Jehol is case-specific and that varied mechanisms were involved. In the case of animal tissues, it is possible that preservation of soft tissues was facilitated by endogenous iron and oxygen from the hemoglobin in blood26,38. The abundant blood vessels in the PFM may explain the high preservation potential of ovarian follicles, although this hypothesis cannot explain why other tissues and organs that are also highly vascularized are not preserved in the same specimens.
In STM10–12 and all other specimens with purported follicles, the overall spherical shape of the follicles is preserved in 2D. Therefore, it is also possible that organic remnants from the original spherical yolk and/or the inner perivitelline membrane are preserved, but extensive further analyses (using different methods such as immunohistochemistry or spectroscopy) are required to confirm this. Additionally, the most external tissue covering preovulatory follicles in extant birds is made of pancytokeratin18, a hydrophobic protein with a high potential for fossilization52. We propose that this hydrophobic molecule may have acted as a barrier and also facilitated the exceptional preservation of follicles. Much more research is necessary to confirm this hypothesis, and more studies on preserved soft-tissues in the Jehol are necessary to further shed light on the modes of tissue preservation through deep-time.
Source: Ecology - nature.com
