Specimen discovery and morphology

Exceptional soft tissues were discovered in an otherwise unprepared Confuciusornis specimen (MES-NJU 57002, Museum of Earth Sciences, Nanjing University) that was collected from the most productive Confuciusornis-bearing layer of the Yixian Formation in the Sihetun area, Liaoning Province, in NE China, by the first author. The exceptional preservation was enabled by taphonomic processes of charcoalification of carcasses by a hot pyroclastic density flow13. Photomicrography, backscatter scanning electron microscopy (BSEM), computed tomography (CT) scanning, X-ray microdiffraction and energy dispersive X-ray spectroscopy (EDX) analyses of the surrounding sediments and long bone mineralizations were described in a prior study13. The specimen bears a suite of morphological characteristics that are diagnostic of Confuciusornis, such as a straight femoral shaft, ball-shaped femoral head with a distinct capital fossa, straight tibiotarsus, proximally fused and very short tarsometatarsus, slit between metatarsal III and IV proximally, metatarsal I attached distally to the lateral side of metatarsal II, slender and splint-shaped metatarsal V, and highly recurved claws with horny sheaths14,15,16 (Supplementary Fig. 1). The proximal and distal tarsals were completely fused to the tibia and metatarsals II–IV, respectively; no sutures are evident in the fully ossified distal tibiotarsus and proximal tarsometatarsus. These skeletal traits indicate that the specimen was skeletally mature17, even though its body size is perhaps slightly small as indicated by the shorter length of its tarsometatarsus compared with previously published specimens (Supplementary Table 1).

Histological sectioning and tissue identification

Two approximately parallel sections oblique to the sagittal plane were prepared from the specimen. Section 1 cuts from the medial surface of the distal tibiotarsus, passing through the medial condyle and a cavity between the tibiotarsus and tarsometatarsus, to the lateral surface of the proximal tarsometatarsus of the right pelvic limb. Section 2 is lateral to section 1, cutting through the medial and lateral condyles, to the lateral surface of the tarsometatarsus (Fig. 1; also see Supplementary Fig. 1b in ref. 13).

Figure 1: Osteology and putative soft tissues of the Confuciusornis hindlimb. Specimen MES-NJU 57002 (right lower limb), showing position of sections. (a,b) Three-dimensional digital images, constructed from microCT scan data, showing approximately dorsal (a) and plantar (b) views of the right distal hindlimb and positions of sections. (c,d) Close-up view of the ankle joint in approximately medial (c) and lateral (d) views. (e–g) Photomicrographs of the ankle joint before sectioning (e), in section 1 (f), and in section 2 (g). (h) Approximately sagittal microCT slice showing positions of horizontal microCT slices. (i–p) Continuous horizontal microCT slices across the distal tibiotarsus (i–l) and proximal tarsometarsus (m–p). cl, condylus lateralis; cm, condylus medialis; crl, crista lateralis; crm, crista medialis; hr, hypotarsal ridge; mt, metatarsal; S1/S2, sections 1/2; sb, spongy (cancellous/trabecular) bone; st, soft tissue; su, sulcus; tib, tibiotarsus; tmt, tarsometatarsus. Scale bar, 5 mm in a–e and g, 500 μm in f, 1 mm in h–p. Full size image

Blackened soft tissues with residual, desiccated linear biological structure are exposed intermittently on the plantar surface of the distal tibiotarsus and then across the ankle joint to the proximal tarsometatarsus (Fig. 1f,g). Scanning electron microscopy-based energy-dispersive X-ray analysis shows that the tissues comprise mainly carbonaceous materials13 (Supplementary Fig. 2). Three types of soft tissues are recognizable.

Tissue type 1 is exposed from the distal tibiotarsus, through the space between the tibiotarsus and tarsometatarsus, to the proximal tarsometatarsus. It is composed of parallel arrays of wavy fibrils forming bundles of 1–10 μm in diameter, separated by linear fissures (‘t/l’, Figs 2a,b and 3; Supplementary Fig. 3). This tissue bears the hallmarks of tendons or ligaments, such as the wavy appearance, parallel arrangement and hierarchical organization of the fibrils18,19, distinct from purely sedimentary features or other tissues. Hence, we interpret tissue type 1 as tendon and/or ligament.

Figure 2: Probable tendons/ligaments and fibrocartilages in the Confuciusornis ankle. The intertarsal joint region of the specimen MES-NJU 57002 (Fig. 1f,g). (a) BSE-SEM image of the square area in Fig. 1f, showing the tarsometatarsus at the top right of the image and the tibiotarsus on the left, with intervening tendon/ligament material (t/l). Within the tendon/ligament, there are regions of fibrocartilage matrix (fc) and cells (arrows) arranged in rows between parallel fibres. (b) Close-up view of the square area in a. (c) BSE-SEM image of the square area in Fig. 1g, showing the tibiotarsus on the left and the tarsometatarsus on the right. A large area of mineralization (m) is visible in the fibrocartilage on the plantar aspect of the ankle joint. (d) Close-up view of the square area in c. Cellular structures (arrows) are embedded in a matrix composed of network of interwoven fibres and smaller mineralizations. (e) BSEM images show that the articular facets of the tarsometatarsus and tibiotarsus in the Confuciusornis specimen contain two thin layers of densely packed, paired cellular structures: an inner calcified layer (yellow arrows) and an outer uncalcified layer (blue arrows). (f) Close-up view of the square area in e. Scale bar, 200 μm in a and c, 100 μm in e, 50 μm in b, 10 μm in d, 20 μm in f. Full size image

Figure 3: Apparent soft tissues in the distal tibiotarsus of the Confuciusornis specimen. Distribution and morphology of the features in specimen MES-NJU 57002 (Fig. 1g). (a,b) Photographic (a) and BSE-SEM image (b) show the blackened soft tissues (dark areas) exposed on the plantar surface of the distal tibiotarsus. Note epoxy may be permeated into the fissures (black areas) in the fossil and between the fossil and the enclosing sediments during sample preparation. (c) BSE-SEM images of the rectangular area in b showing structures of tendons/ligaments and fibrocartilages (insets). (d) Close-up view of the tendons/ligaments in c. See Figs 1 and 2 for abbreviations. Scale bar, 100 μm in a and b, 20 μm in c and 2 μm in d. Full size image

Tissue type 2, with thickness up to 0.5 mm, occurs in the areas on the distal tibiotarsus and the proximal tarsometatarsus where the tendons/ligaments would have wrapped around the condyle and cotyle. It consists of oval or round cellular structures 5–10 μm in diameter that are either embedded in a matrix composed of a network of interwoven fibrils and tiny mineralized zones, or arranged in rows between parallel fibres (‘fc’, Figs 2 and 3a–c). These features are typical of fibrocartilage20,21, and hence we interpret this tissue’s identity as fibrocartilage. The fibrocartilages appear to be locally (at least partly) ossified, as indicated by the presence of small mineral precipitates in the matrix and mineralized cellular structures on the upper surface of the underlying bone (‘m’, Figs 2c,d and 3a–c).

Tissue type 3 is preserved along both articular facets of the tarsometatarsus and tibiotarsus. It contains two thin layers of densely packed, paired cellular structures in a matrix composed of a network of interwoven fibrils: an inner mineralized layer and an outer unmineralized layer (Fig. 2e,f). The cellular structures are oval or round and about 10 μm in diameter. The structures, resembling the lacunae left by paired cells of articular cartilage in their shape, size and positions of occurrence22, indicate that the articular cartilages are partly preserved. The mineralized and unmineralized layers correspond respectively to the calcified and uncalcified zones of articular cartilages. This interpretation is supported by gross morphology and histology of the corresponding extant avian tissues (Supplementary Figs 4,5 and 6), and previous reports of cartilage preservation in fossils23.

Chemical analyses of possible soft tissues

Tendons, ligaments and cartilage are mainly composed of collagen and the proteoglycan aggrecan24. Survival and detection of residual amide functional groups derived from precursor proteins within fossil specimens is well documented via Fourier transform infrared (FTIR) and time-of-flight–secondary ion mass spectrometry (ToF–SIMS)5,6,7,8,9,10,11,25. We tested our inferences based on the morphological similarities of these putative soft tissues to tendons, ligaments and fibrocartilages with chemical analyses.

FTIR analysis was applied to the tissues exposed on the polished section in reflection mode and the spectra were compared with those of modern intact collagen and aggrecan26. As shown in Fig. 4a, the fossilized soft tissues have three main strong absorbance regions. The region of 960–1,160 cm−1 is strongest with a prominent peak at 1,033 cm−1, followed by that of 1,400–1,700 cm−1 with two strong peaks at 1,510 and 1,652 cm−1, respectively. The region of 1,200–1,300 cm−1 is relatively weaker, with a peak at 1,248 cm−1. The peaks at 1,652 and 1,510 cm−1 correspond to the diagnostic amide I and II absorbances, respectively, which appear distinctly in the spectra of collagen and aggrecan. The presence of the amide I peak at 1,652 cm−1 in both fossil spectra, combined with the additional structure in both spectra from 1,652 to 1,400 cm−1, indicate that organic material is present within the fossil specimen and that products from the breakdown of proteinaceous material most likely contributed to this organic matter. The peak at 1,033 cm−1 presumably is a Si-O stretch mode from microcrystallites of a silicate phase within the fossil. Absorption due to the amide II band, at 1,550 cm−1 in the reference spectra, may be present within the broad elevated region of absorbance in the fossil tissue, and may even be slightly shifted to lower wavenumbers and thus contribute to the peak visible at ∼1,510 cm−1. At 1,248 cm−1 in the fossil specimen, the broad peak would be consistent with amide III but may be convolved with sulfate as seen in the aggrecan spectrum. This region (1,400–1,700 cm−1) that includes the amide I and amide II peaks provides the most prominent FTIR absorption peaks of collagen from modern cartilage26,27. FTIR mapping shows that the areas of this strong absorbance correlate with those of the putative tendons/ligaments and fibrocartilages (areas 2, 3, 5–7 in Fig. 4e,f), although they also overlap the areas of fissures (the black areas in Figs 3a–c and 4e) where the signal probably is interfered with signal from epoxy. These FTIR spectra and mapping imply that amino acid residues may be present.

Figure 4: Chemical analyses of the putative soft tissues in the Confuciusornis hindlimb. (a) Comparison of FTIR spectra (1–2) obtained from two spots on the supposed soft tissues with the typical proteoglycan aggrecan (3) and collagen (4)26. (b) Mass spectra of detailed region in 274–276.5 AMU of the supposed soft tissues. (c) Sulfur K-edge XANES spectrum from the high-sulfur region showing that the dominant sulfur species is sulfate (edge position for sulfate standard at 2,482 eV indicated by vertical dashed line). (d) False colour synchrotron microfocus XRF map of sulfur in the rectangular area in Fig. 1g shows that the high-sulfur regions (yellow-green colour, inset:#1–6) are correlated with the distribution of tendons/ligaments (#1–6). Scale bar, 100 μm. (e–j) Comparison of the distribution of the inferred fibrocartilages (#2–3, 5–7) and possibly mineralized fibrocartilages (#1, 4) in BSE-SEM image of the rectangular area in Fig. 3b (e) and FTIR map of the strong absorbance region of 1,400–1,700 cm−1 in the same area (f) and ion images of the spatial signal intensity distribution in the square area in Fig. 4e for Al+ (g), Fe+ (h), NH 4 + (i) and the peaks at 275.16 AMU (j) in negative ToF–SIMS spectra. The FTIR map was collected using a Thermo IN10MX infrared microscope with a cooled MCT detector. Full size image

ToF–SIMS with imaging capability was applied to putative soft tissues on the polished section (the square area in Fig. 4e). Spectrum and image data were acquired in the bunched mode (m/Δm ∼6,000) at a spatial resolution of ∼5 μm at analysis depths up to 1 and 1.7 nm, respectively. The spectra presented in Fig. 4b and Supplementary Fig. 7f–j were obtained at analysis depth up to 1 nm from the areas enclosed by the blue line in Supplementary Fig. 7a,b, which mainly consist of the inferred soft tissues. Ion images reveal that the spatial signal intensity distribution for peaks at 275.16, 293.17 and 413.26 AMU in negative ToF–SIMS spectra highly correlates with the distribution of the inferred fibrocartilages (Fig. 4i; Supplementary Fig. 7e,k), and in contrast to those for NH 4 + (Fig. 4j) and Al+ (Fig. 4g) in positive ToF–SIMS spectra, which superimpose with the distribution of fissures that may contain epoxy and the enclosing sediments, respectively. Spatial signal intensity distribution of mass values for peaks at 277.15 AMU (Supplementary Fig. 7c) and 415.26 AMU (Supplementary Fig. 7d) in positive ToF–SIMS spectra also weakly superimposes on the distribution of the inferred soft tissues, but is interfered with by the signal from the sedimentary matrix and probably epoxy in the fissures. Ion images acquired at analysis depth up to 1.7 nm (Supplementary Fig. 7i–t) are consistent with those at analysis depth up to 1 nm. This rules out the possibility that spectra resulted from outermost surface contamination.

A characteristic feature of the secondary ion spectrum of an organic molecule M is the appearance of the quasi-molecular ions (M+H)+ (M+cation)+, and (M−H)− (ref. 28). The fragment ions of 275.16 and 293.17 AMU (=275.17+1H 2 O) in negative spectrum and 277.15 AMU in positive spectrum probably represent a similar molecule with a mass of 276.16 AMU. Likewise, the measured peaks at 413.26 AMU in negative spectrum and 415.26 AMU in positive spectrum possibly represent a similar molecule with a mass of 414.26 AMU. There are many candidate molecules with the measured ToF–SIMS peak masses and we cannot resolve definitive sequence assignments, because all we have is mass data. Considering that proteins may be degraded into peptides of various sizes in variable states of protonation/deprotonation, the measured mass peaks were tentatively interpreted by comparing probable peptide sequences with the peaks resolved via ToF–SIMS. The 276.16 AMU peptide needed to explain the 275.16 negative peak and 277.16 positive peak could possibly be a Gly-X-Y tripeptide (for example, Gly-Ser-Asn or Gly-Ser-Hyp). The mapped distribution of this mass fragment indicates that it is indeed derived from regions within the fossil that display the presence of amino acids. Because Gly-X-Hyp is one of the most common sequences in collagen, the distribution of this proposed moiety (Fig. 4j; Supplementary Fig. 7) is consistent with the presence of collagen residue as inferred from the FTIR data. The required peptide mass at 414.26 AMU (to explain the peak at 413.26 AMU in the negative spectrum and 415.26 AMU in the positive spectrum) is more problematic. Although it could represent protein-derived residue, given its distribution we do not base any conclusions on this resolved mass. However, we conclude that the ∼276 AMU data are consistent with the presence of Gly-X-Y residues. Therefore, the ToF–SIMS maps and spectra strengthen the inference from the FTIR spectra that amino acid residues from collagen may be present in the fibrocartilage zones of this specimen.

X-ray chemical analyses

Connective tissues such as cartilage or tendons/ligaments normally have high-sulfur content and their dominant sulfur species is sulfate. Synchrotron rapid-scanning X-ray fluorescence mapping revealed that the sulfur content of the soft tissues was higher than the overlying bone, the embedding sediments and the fissures between them (Fig. 4d). Subsequent X-ray absorption near-edge spectroscopy showed that the sulfur speciation in this region of the fossil is dominated by sulfate (Fig. 4c). This is wholly consistent with the FTIR peak at 1,248 cm−1, which relates to an overlapping absorbance region of Amide III from collagen and sulfate from aggrecan. Since the region of high sulfur was confined to the soft tissues, the sulfur may derive from original sulfate or the breakdown of original organosulfur compounds.

Summary of chemical analyses

These chemical analyses support our inference that the segmented biological structures probably are the preserved residues of tendons/ligaments and cartilages. Interestingly, an ion image of spatial signal intensity distribution for Fe+ (Fig. 4h) in the positive ToF–SIMS spectra can also be directly superimposed onto the inferred distribution of soft tissues, which supports the hypothesis that iron may be involved in the preservation of soft tissue5,11.

Morphological interpretations of likely soft tissues

The deepest tendons that cross the plantar surface of the lower leg in extant birds are parts of the digital flexor muscles4,29,30. This implies that the preserved residual tendons/ligaments are remnants of those digital flexor tendons, and intertarsal ligaments closely associated with them. The tendons/ligaments pass through two cartilaginous structures, a sulcus on the distal tibiotarsus and a ridge on the proximal end of the tarsometatarsus.

The sulcus is delimited by two cristae against the condyles on the distal tibiotarsus. The cristae, about 1 mm thick, are porous and locally fragmented. Their lower parts are fused to the underlying bone, and their upper surfaces are irregular and covered by partly mineralized fibrocartilages in the area contacting with tendons/ligaments (Figs 1e–l and 3a,b). Partly intact preservation of spongy condyles and continuous cortical bone across the cristae and sulcus (Fig. 1i–l) indicate that the cristae are not derived from compression or displaced cortical bone, even though the bone apparently underwent some compression during fossilization. The fibrocartilaginous nature of the cristae is supported by the distinct light grey colour of the cristae in comparison with the brown colour of the remaining bone exposed in a further two Confuciusornis specimens from the Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences (IVPP; IVPP 18156 and IVPP 13175; Supplementary Fig. 6). The sulcus and the contacting fibrocartilage anatomically correlate with the caudal intercondylar sulcus and the tibial cartilage that is situated in the sulcus in extant birds29,30.

The ridge on the proximal end of the tarsometatarsus is composed of fibrocartilage and a crescent-shaped mineralization ∼0.1 mm thick on its plantar side (Figs 1g, 2c,d and 5). It is elevated and inflated in its medial to central position. Distal to the ridges, sulci are formed on the central to lateral position of the proximal tarsometatarsus, which connect to the grooves against metatarsals II, III and IV in the area where the metatarsals are not fused (Fig. 1m–p). Such a cartilaginous ridge with sulci distal to it can be observed in other Confuciusornis specimens (for example, IVPP18156 and IVPP 18168; Supplementary Fig. 6). The ridge occurs at the position of the hypotarsus in ornithuromorph birds31, but it is less distinct and more cartilaginous compared with the latter structure, even with those seen in early ornithuromorph birds, such as the Late Cretaceous Patagopteryx, Pengornis, Yixianornis, Apsaravis and Ichthyornis, which have a flat bony projection or unprojected discrete surface without canals and sulci32,33,34. In contrast, the ridge resembles an intermediate state of the hypotarsus in the ontogeny of extant birds, in which the hypotarsus remains cartilaginous until the latest stages or after hatching, when it ossifies from a separate centre located on its distal medial corner35,36.