The microscopic organisation of the epidermal remains in YFGP-T5199 closely resembles decayed keratin fibrils and eumelanosomes found in extant bird feathers, especially after selective biodegradation of the amorphous polymer matrix27,28,30,31. The exceptional morphological fidelity of these filamentous appendages also reveals a fibrillar hierarchy reminiscent of the rachis and barb cortex27, which may have imparted a flexural stiffness to the ‘forecrown’ feathers in YFGP-T5199. The mechanical architecture of paravian feathers can thus be shown to extend beyond gross macroanatomy36,37, to a sub-cellular level of biological organisation.

Multiple independent lines of evidence advocate a eumelanosome origin for the rod-shaped microbodies in the epidermal tissues: (1) the presence of animal eumelanin; (2) the size, shape, distribution and parallel alignment, which unlike bacterial cells do not form serial chains indicative of microbe fission (see ref. 15, figs 1e and 2a, S1c2 and ref. 30, fig. 6); and (3) the embedment within fibril-like structures similar to feather keratin. Yet despite this striking morphological resemblance, we failed to detect any proteinaceous components indicative of keratins. Instead, the fibrous tissues consisted of eumelanin and calcium phosphate, the latter possibly derived from mineral replacement. Indeed, post-burial melanin leakage might have provided a mechanism for either epidermal tissue stabilisation or replication in YFGP-T5199 that was further facilitated by the rapid growth of authigenic minerals38,39,40. Early mineralisation is a common way of preserving labile soft tissues38,39,40 and often involves calcium phosphate in fossilised melanic and/or keratinous structures, including ink sacs41,42, feathers5,43 and claw sheath material44.

The association of biomolecules with a mineral substrate is thought to increase the preservation potential of organic compounds, either via adsorptive inhibition of autolysis in decay-inducing enzymes, or by fixation of mineral ions into stabile organometallic complexes that impede molecular breakdown44,45. Polymer-calcium phosphate39,40,44 and/or polymer-clay38,45,46,47 interactions could thus be responsible for the retention of eumelanin molecules in YFGP-T5199. Regardless, eumelanin itself is resistant to decay48 because of its extremely dense and insoluble polymer composition that is both antimicrobial and chemically robust49,50, even in comparison with keratin9. Furthermore, mature melanosomes are essentially solid aggregations of melanin, which is polymerised onto an insoluble amyloid fibril scaffolding51,52, thus imparting an architectural stability that likely allows these specialised organelles to persist in the fossil record.

Some imprints observed in the matrix bordering the fibrous microstructures (e.g., A5) are reasonably interpreted as external moulds of pigment organelles based on their dimensional, distributional and chemical compatibility with the rod-like microbodies interpreted here as remnant eumelanosomes (Fig. 5 and Supplementary Fig. S2d–f) and might have been derived via sample preparation or disruption – e.g., impression from microbodies located on the counterpart (a scenario experimentally shown to produce melanosome imprints in the keratin matrix of modern feathers8).

A melanosome origin is also plausibly inferred for other mouldic structures in YFGP-T5199, including those exposed in A6 (note the continuous alignment of the impressions in A5 and A6; Fig. 5b). However, the molecular composition in this region broadly corresponds to that of the host rock (albeit with an increased amount of calcium phosphate), indicating mould formation primarily by aluminosilicate clays. It is therefore possible that local, rapid nucleation and precipitation of clay minerals proceeded in synchrony with the decomposition of the keratinous feather material, thereby encapsulating more stabile organic structures (such as the eumelanosomes) within clay nanofabrics. At a later stage, the entombed organelles also decayed, leaving hollow void spaces that for unknown reasons remained empty (assuming that these melanosome ‘pseudomorphs’ are not negatives from positive reliefs on the counterpart slab).

The affinity of other imprints is more enigmatic, including those in A7 (Supplementary Fig. S2h). Not only are these voids morphologically more diverse, but they are also highly disorganised in comparison to the solid eumelanosomes (Fig. 2a and Supplementary Fig. S2h). Spatially, these impressions are also seemingly restricted to aluminosilicate clays and microcrystalline clay aggregates along the bedding plane (Fig. 2a and Supplementary Fig. S2h). We were unable to locate any three-dimensional microstructures matching the imprints, implying that maker(s) of these moulds were less resistant to decay than the eumelanosomes.

In modern feathers, melanosomes can be organised into discrete layers where individual organelles are either consistently aligned or more erratically oriented with only local uniformity in directions8,53. Thus, the fossil imprints may comprise moulds of melanosomes that exhibit more variability in shape and originated from the outer cortex (which can exhibit less melanosome alignment8). These organelles may also be more prone to degradation because of greater exposure. Alternatively, preservational biases and diagenesis could potentially modify the appearance of fossil structures46,54, although this would require the pigment organelles to be transformed not only in size (as has been previously demonstrated26), but also in shape and orientation during the fossilisation process (assuming that they were originally aligned in approximate parallel with the bedding plane).

On the other hand, colonies of keratin-degrading microorganisms often comprise a consortium of taxa, resulting in a mixture of shapes and sizes30. They are also inherently associated with decaying feathers and can form clustering patterns where neighbouring cells are oriented in common directions (see ref. 30, Fig. 6). Furthermore, because some decay is necessary to initiate mineralisation39 and because calcium phosphate precipitation can be microbially induced40,55, a microorganismal mediator could be rationally invoked in the partial replacement of the feather material. This, coupled with morphological evidence of fossilised microbes56, has implications for the interpretation of the more enigmatic imprints observed in the matrix bordering the fibrous microstructures in YFGP-T5199 (Fig. 2a and Supplementary Fig. S2h). Given the extensive fossil record of microorganisms56,57 and that some of these voids were not associated with an animal eumelanin molecular signature (e.g., A7), a microbial origin for these particular imprints cannot be excluded. Accordingly, we argue caution against interpreting all fossilised microbodies and impressions as melanosomes and reconstructing plumage colours based on morphology alone.

An interesting aspect of the fossilised melanosomes in YFGP-T5199 is their exclusively elongate shape (Fig. 2b and Supplementary Fig. S2e). This deviates markedly from the relatively stocky microbodies reported from the ‘forecrown’ feathers of another referred specimen of A. huxleyi (BMNHC PH828, housed in Beijing Museum of Natural History)5. With a few possible exceptions (Fig. 2a—arrowhead), we also detected no pheomelanosome-like structures (see ref. 5). Several explanations might account for these discrepancies, including ontogeny, intraspecific variability and sexual dimorphism, as well as taphonomy, and/or sampling (that is, different regions within a multi-coloured crest). We also have to entertain the possibility that: (1) YFGP-T5199 and BMNHC PH828 represent different taxa; (2) integumentary melanosomes intermingle with melanosomes from other parts of the body in one of the two specimens; and (3) the microbodies and impressions reported by Li et al.5 and us are different structures altogether.

Indeed, BMNHC PH828 is considerably smaller (~60% by ulna and tibiotarsus length) than YFGP-T5199 and hence it may represent an earlier ontogenetic stage of A. huxleyi. Furthermore, YFGP-T5199 possesses a uniquely short dorsodistal process of the ischium (see the Supplementary Methods section online), which suggests the possibility of intraspecific and/or sexual differences that may also be reflected in the expressed colour pattern. However, the referral of BMNHC PH828 to A. huxleyi is problematic and we cannot exclude that this specimen represents a closely related but different paravian taxon (see the Supplementary Methods section online).

Additionally, while it has been shown that increased temperature and pressure can reduce the size of melanosomes26, such alterations may not also include shape (but also see ref. 10). Taphonomy, therefore, presumably cannot account for the different microstructures seen in YFGP-T5199 and BMNHC PH828. Even though melanosomes can potentially disperse during decay58, the microbodies in S1 are located deeply within a fibrous matrix interpreted as fossilised keratin. We thus conclude that the melanosomes most feasibly derive from the ‘forecrown’ feathers of YFGP-T5199 as opposed to other dermal tissues and/or internal organs (but whether this is also true for BMNHC PH828 has yet to be determined).

Although a re-investigation of the affinity and preservation of BMNHC PH828 is beyond the scope of this study, we note that the ‘pheomelanosome’ imprints reported by Li et al. (ref. 5, Fig. S5) are preserved in sedimentary grains rather than recognisable feather traces, similar to our more enigmatic impressions. Thus, these imprints may also represent either remobilised melanosomes or non-melanosome microstructures.

Our integrated structural and direct chemical approach provides compelling evidence that eumelanosomes and endogenous eumelanin pigment are preserved in the feather remains of YFGP-T5199. This result adds to a growing chronicle of molecular eumelanin detection in fossils33,35,42,59,60 and demonstrates the aptitude of rigorous experimental techniques for identifying ancient biomolecules and their use in characterising ‘palaeo-colours’.