We evaluate statements used by others to support a melanosome origin for microbodies in fossil feathers and put forth alternative interpretations equally supported by the same data. (1.) Localization of microbodies to ‘dark’ and absence from ‘light’ regions of one fossil feather support a melanosome origin4. Three hypotheses exist to explain the striped patterning of the fossil feather reported in Vinther et al. 2008. First, the original feather, in life, was also striped, with melanosomes distributed in the colored regions of the feather and no melanosomes in the uncolored parts and the fossils preserve this original pattern. Second, microbial overgrowth on the surface of the feather is distributed according to the relatively more nutrient-rich pigmented feather regions over unpigmented areas36. Third, microbes preferentially colonized and completely degraded those feather regions most easily broken down (e.g. unpigmented)37,38; thus were no longer present in these regions during fossilization, but continued to act on the more resistant, melanin-containing regions. (2.) Densely packed and aligned/organized layers support a melanosome origin ( Fig. 2c–e )7,13. Recently published research indicates original melanosome geometry and distribution are altered with heat and pressure31, diagenetic processes affecting fossils and that have not been taken into account in previous research claiming melanosome morphology in fossil feathers reflects their original color.

Data presented here show microbial overgrowth is dense and can be aligned, whereas internal melanosomes are more sparsely distributed and relatively random in orientation as observed in both SEM and TEM. Our data show this description is more consistent with biofilm overgrowth (Fig. 2a, b) than melanosomes (Fig. 1b and c). In addition, bacteria can align (Fig. 1e, 2a and Supplementary Fig. S1c2) and follow the contours of a feather (Supplementary Fig. S2) in layers. Unlike most bacteria, which usually exhibit more uniform and smooth cell surfaces39,40, melanosomes, as observed in a previous study26, are not smooth, but rather have bumpy and non-uniform surfaces (Fig. 3b and d). Because bacteria have a tough cell wall external to the plasma membrane41, this granular topography26 may be more difficult to observe in melanin-containing bacteria than in eukaryotic melanosomes. This remains to be tested. (3.) Elements consistent with melanin, including Ca2+, Cu2+and Zn2,43,44, are associated with feathers and thus support a melanosome origin. However, these biomarkers are also used and/or sequestered by bacteria45,46, including common soil bacteria and other microorganisms and are also part of the sedimentary environment. These microorganisms are also capable of synthesizing melanin29, thus elemental data alone cannot be used to discriminate microbes from melanosomes.

Our examination of the fossil feather ascribed to Gansus yumenensis showed no microbodies of any type, but did reveal ‘mouldic impressions’ (Fig. 6) similar to those described in previous studies. However, these impressions did not vary between black and brown regions of the feather and were also observed on sediment grains (Fig. 6) (confirmed with EDS data) associated with the fossil. Therefore, because sediment grains do not contain melanosomes, it more parsimonious to propose these ‘mouldic impressions’ represent a microbial origin (remnant EPS) than intracellular structures derived from the original feather. A very similar image was presented in Barden et al. 2010 (Fig. 1H) but elemental data were not mapped, so identification as a sediment grain cannot be confirmed in their paper. Visual, textural (Fig. 5 and Fig. S4) and elemental data (Fig. 7 and 8 and Table 1) from the fossil feather suggest differential diagenetic processes acting on the different regions of the feather. The reason for these differences is beyond the scope of this paper and requires additional studies.

Pending the identification of definitive molecular or chemical signals unique to either melanosomes or microbes in extant feathers that are likely to persist across geological time, distinguishing microbes from melanosomes in fossils may be difficult. Until new data are presented, we propose the following criteria to support a melanosome origin for microbodies associated with fossil feathers: 1) a taphonomic mechanism must be demonstrated for removing resistant keratin while leaving the intracellular organelles intact; 2) electron-dense material should be localized to the microbodies using TEM-EELS (TEM coupled with energy loss spectroscopy) or TEM-EDX; 3) melanosome-specific (e.g. cargo proteins5) or bacteria-specific (e.g., peptidoglycans16) biomolecules should be localized to the structures to eliminate the alternative, using in situ surface techniques (e.g., time of flight secondary ion mass spectrometry (TOF-SIMS42)), or other softer mass spectrometry imaging methods.

The ‘mouldic impressions’ described in fossil feathers imply that the microbodies were once present and subsequently degraded from an amorphous material that retained the impression through fossilization. Therefore, if the structures are melanosomes, this material should resemble β-keratin, because the β-keratin matrix of feathers is highly resistant to degradation47. β-keratin is a rigid structural protein comprising ~80% of the organic matrix of mature feathers48. Its multiple cross-links, twisted-pleated-sheet tertiary structure and hydrophobic amino acid composition49 confer high preservation potential to structures comprised of this protein50. Mammals do not produce β-keratin51, thus common contamination by human keratins can be easily recognized. If microbial, the material should retain microbial-specific biomarkers in the environment immediately surrounding these microbodies.

Some papers state the keratin matrix is completely degraded in fossil feathers and no feather structure remains4,9,11,13, but fail to state what material, then, retains ‘mouldic impressions’. If feather structure is not preserved, how is the object identifiable as a fossil feather and how has a biofilm source been eliminated? The presence or absence of keratin has not been tested in any fossil feather purporting to contain melanosomes. Yet, the melanosome hypothesis posits that melanosomes are preserved ‘in life position’. Modern feather melanosomes (‘in life’) are always embedded in a keratinous matrix, thus the ‘mouldic impressions’ cited by many in support of the melanosome hypothesis are assumed to be made in the original keratinous matrix of the feather, an assumption that has never been tested.

It should be noted that handling history and full depositional description are often not included in studies purporting to recover fossil melanosomes. Excavation of a fossil feather as part and counterpart could be interpreted differently than a feather collected as a whole specimen. This information is critical for determining where these microbodies are localized (ie. inside versus on the surface of the feather).

More importantly, even if irrefutable data support a melanosome origin for microbodies in a given fossil, imparting color to the entire organism, or even the entire feather, based upon their presence cannot be inferred. All melanized feathers in extant birds contain both eumelanin and phaeomelanin6; it is the relative abundances of these two melanins that determine the expressed color of a pigmented feather6. Claiming a ‘red-orange’ or ‘black-grey’ color for entire fossil organisms based upon identification of round or elongate morphologies is overly simplistic, because in living birds, pigment molecules of multiple types are employed to confer hues of brown, red, orange, etc.6. Coloration of feathers is complex, the result of expression of more than one type of pigment (e.g. porphyrins, carotenoids)6, which may be more labile, with lower preservation potential, than the relatively resistant melanins. Without preservation of all pigments originally employed, original organismal color cannot be interpreted with accuracy.

The initially proposed hypothesis of a microbial origin1 for these microstructures observed in multiple fossilized feathers, as well as other fossil material from the Messel deposits2,3 has not been refuted, or indeed addressed, with data presented in previous studies, but is supported by the data we present herein. The present data do not support the melanosome hypothesis for these fossilized microstructures. Morphology alone is insufficient to distinguish between a melanosome and/or microbial origin, but data that capitalize on distinct chemical differences between melanosomes and microbes are needed to support one hypothesis over the other. With the exception of one fossil feather study where the chemical data are not of high resolution32, the only in-depth chemical data presented for microbodies in the fossil record that seem to support a melanosome origin are derived from marine42,52,53,54 rather than terrestrial environments, where preservational conditions are very different than the lacustrine environments from which most feathers have been recovered1. Additionally, geochemical data from fossil feather ‘melanosomes’ are compared only with that derived from extant melanosomes; microbes are not included in the comparative data50.

Furthermore, because the shape of melanosomes has been used to interpret color4,7,8,9,10,11 and behavior7,11 in extinct animals, distinguishing melanosomes from microbes is critical to acceptance or rejection of these hypotheses. As McGraw warned “[…] it is wise to withhold classification of a color as partially or wholly melanin-based before the appropriate biochemical tests are conducted”6. How much more should this caution be applied to extinct organisms?