Evidence corroborates identity of isolated fossil feather as a wing covert of Archaeopteryx

The historic fossil feather from the Jurassic Solnhofen has played a pivotal but controversial role in our evolutionary understanding of dinosaurs and birds. Recently, a study confirmed the diagnostic morphology of the feather’s original calamus, but nonetheless challenged the proposed identity as an Archaeopteryx covert. However, there are errors in the results and interpretations presented. Here we show that the feather is most likely an upper major primary covert, based on its long calamus (23.3% total length) and eight other anatomical attributes. Critically, this hypothesis is independently supported by evidence of similar primary coverts in multiple specimens of Archaeopteryx–including from the same fossil site and horizon as the isolated feather. We also provide additional insights, such as an updated colour reconstruction of the entire feather as matte black, with 90% probability. Given the isolated nature of the fossil feather, we can never know the anatomical and taxonomic provenance with 100% certainty. However, based on all available evidence, the most empirical and parsimonious conclusion is that this feather represents a primary covert from the ancient wing of Archaeopteryx.


Supplementary
Thus, in addition to the initially faint calamus of the isolated fossil feather, it is also possible that the calamus outline faded shortly after von Meyer's illustration. This is plausible given that von Meyer had received the feather specimen directly from the quarry 8 . It is unknown when exactly von Meyer made his illustration, which occurred sometime between the fossil's discovery in spring or early summer 1861, and the publication in April 1862 12,13 , although no illustration or mention thereof is made in von Meyer's letters from August and September 1861 14,15 . Regardless, by the time de Beer (1954) 16 published photographs of the two feather slabs, no such calamus was visible on either. Degradation of any visible calamus was likely exacerbated by exposure and handling since its discovery (e.g., note the fingerprints on the Munich slab's feather 6,17 ). Some decades later, the Berlin slab ( Supplementary Fig. S1) was further prepared, and any visible calamus traces that remained may have been removed at this time. Indeed, Griffiths (1996) 18 explicitly mentions damage to the fossil due to "poor preparatory technique," and scratch lines are clearly visible on the calamus region in his Plate 5, Fig. 1 "There are several dimensions of feathers, the measurements of which can help to identify them as to the kind of bird or the tract where they originated. One or more of these measurements is included in each of the descriptions of feathers in this chapter. The dimensions, as we have measured them, are as follows: Length.-The length is measured from the bottom of the calamus to the tip of the vanes, with the shaft as straight as possible and the terminal barbs extended as far as possible.
Width.-The width is the maximum distance across both vanes with the barbs at their normal angles from the rachis. In flight feathers and their coverts, it may be necessary to measure the widths of the vanes separately.
Calamus length.-The length of the calamus is measured from the rim of the superior umbilicus, as close as possible to the midventral line (depending on the structure of the afterfeather), to the proximal end." Given that the superior umbilicus is not visible in the fossil, the lowermost barbs were used as the distal limit of the calamus (Ibid., p.235): "The division between the calamus and the rachis is marked by the lowermost barbs and by a small opening to the calamus, the superior umbilicus (umbilicus superior, umbiliciform pit) on the ventral surface of the shaft ( fig. 159)." Relative calamus length refers to calamus length divided by total feather length (see also 15. Anatomical attributes). To account for the curvature in the isolated fossil feather and replicate the aforementioned measurements "with the shaft as straight as possible", the curvilinear lengths of the calamus and rachis were used-as if the fossil were a modern feather that could be straightened out. Some additional notes on the three landmarks used for these measurements: Fig. S11a-d). In von Meyer's two descriptions of the feather, he noted that "The end of the vane is somewhat obtusely angled." 14 , and that this distal tip had a "blunted end" that was "less rounded, more angularly formed than in the living Grey Partridge." 8 However, he drew the distal tip (a) as somewhat more rounded than it appears in the two slabs. In the Berlin slab (b), some damage is visible at the barb edges, due to the aforementioned preparatory scratches 17,18 (Griffiths 1996: Plate 6 18 ). In the Munich slab (d), the feather shows a slightly sharper distal leading edge and tip than in the Berlin slab. Fig. S11e-h). The feather on the Berlin slab was coated with varnish, which artificially thickened the appearance of barbs at the base 17 (f). Fig. S11i-l). Given that the proximal portion of the calamus is not visible in the present-day Berlin and Munich slabs, the only evidence of its morphology is von Meyer's original mirror trace 8 and the recent laser-stimulated fluorescence (LSF) image 6 . In the former, no distinct border was drawn for the proximal tip (j). As von Meyer noted, "The feather is excellently preserved; only the end of the quill is less clearly expressed" 8 . The proximal tip is indistinct in the LSF image as well (l). Albeit speculative, the light grey region immediately below our reconstructed calamus (k) suggests the possibility that the proximal tip may have been slightly longer, perhaps representing a more gradual tapering to the inferior umbilicus. If so, our current measurement of relative calamus length would be an underestimation. 15. Anatomical attributes. See schematic in Fig. 2b. Table 1 includes designations that are generalized to extant birds, whereas Tables 2 and S1 include designations specific to three Archaeopteryx specimens. Attributes are assessed independently within a tract. Therefore, not all designations for a tract may apply to a single feather, and a single designation may not apply to all feathers within a tract. Both circumstances are particularly true in the case of "maybe" (occasionally). Numerous "maybe" designations thus have a multiplicative effect, further increasing the improbability of such tracts as a potential match for the isolated fossil feather (e.g., primaries in Table 1). Fig. 2a and Supplementary Fig. S12 serve to quantitatively assess the relative calamus lengths across feather tracts in one extant specimen, and are not meant to be interpreted as strict absolute limits across all of avian diversity. Specifically, after the relative calamus lengths of the UMPC tract, those of the primary feathers (n = 10, range = 11.1-18.5%; dark grey) are the next closest match to that of the isolated feather (23.3%). In certain species, the relative calamus lengths of primaries can be as long as that of the isolated feather, or even too long. For example, in large flying birds such as swans, the relative calamus length of distal primaries can exceed 30% 10 . Therefore, in Table 1 we designate the relative calamus length for primaries as "maybe". All other feather tracts are considered ruled out by the relatively long calamus of the isolated feather (Fig. 2a) S5b) and state that these coverts "can be distinguished from the upper major primary coverts by the shorter calamus." Thus, we designate the uMPC tract as "no". The calamus-rachis demarcation was not observable in any of the major primary coverts preserved in the skeletal specimens of Archaeopteryx.

#1. Relative calamus length refers to calamus length divided by total feather length (see also 12. Calamus measurements). Values from
#2. Length refers to total feather length, from the proximal tip of the calamus to the distal tip of the vanes ("with the shaft as straight as possible and the terminal barbs extended as far as possible" 10 ). It should be noted that there are exceptions to the general rule of certain modern and Archaeopteryx feather tracts being too long (compared with the isolated feather), often with exclusionary tradeoffs. For example, "maybe" is designated for the Archaeopteryx distal primaries and secondaries (Table S1), given the few distalmost and proximalmost feathers being exceptionally shorter than the rest of the respective tracts (Fig. 4). However, for each of these particular feathers of appropriate length, the width and/or vane asymmetry is uniquely inconsistent with that of the isolated feather.
#3. Width refers to the maximum distance across both vanes. This is a relatively uninformative attribute, given that the width of the isolated feather is potentially consistent with that of all nine tracts of modern feathers. This attribute does however exclude the narrowvaned contours observable in the Altmühl and Berlin specimens of Archaeopteryx.
#4. Aspect ratio refers to an individual feather's length (#2) divided by width (#3). Given that all three attributes are assessed independently, tracts (and/or individual feathers) may be designated "no" for one attribute but "yes" for another. For example, Table S1 contains three tracts designated "no" for aspect ratio, despite designations of "maybe" for length and "yes" for width. In total, aspect ratio eliminates six tracts in Archaeopteryx, including the primaries and secondaries.
#5. Lateral curvature refers to a posterior curve of the centerline, towards the feather's trailing edge (not to be confused with the animal's anatomical "lateral"). This is differentiated from an S-shaped centerline, which has an inflection point that curves the rachis towards the feather's leading edge. As discussed by Norberg 20,21 , flight feathers are curved backwards to increase dorsoventral stiffening, and to permit nose-up rotation during upstroke in order to let air through the wing.
#6. Barb angle refers to the angle between a barb and its rachis. Given the vast amount of variation in barb angles among extant bird species, the presence of barb angle asymmetry was used to assess consistency with the isolated feather (i.e., more acute barb-rachis angles on the leading vane compared with those on the trailing vane 22 ). Given the more tractable sample size of Archaeopteryx specimens used in Tables 2 and S1, both the presence of asymmetry and measurements of barb angles were used to assess consistency with the isolated feather (see also Supplementary Figure S15). Functionally, barb angle in part determines the vane width and flexibility 10,23 .
#7. Vane asymmetry refers to the narrower width of the leading vane compared with that of the trailing vane 10 . Vane asymmetry in the isolated feather was mentioned by von Meyer in his initial description 14 , and measured by Speakman & Thomson 1994 24 as 2.2 at 25% distance from the feather tip. In modern birds, vane asymmetry is usually found in flight feathers and their coverts, and in some tail feathers 10,25 . This attribute is associated with aerodynamic function, by stiffening the narrower leading vane 25 , and enabling rotation about the longitudinal feather axis during flapping flight 20,21 .
#8. Vane closure refers to the closed pennaceous vanes that are provided by interlocking barbules (e.g., Supplementary Fig. S16), and that allow for feathers to function as a coherent aerodynamic surface 10,22,26 .
#9. Angled distal tip refers to the leading and trailing edges of the vanes meeting at an angle, as opposed to forming an entirely rounded tip. Previous authors have also referred to the former condition as "blunted" 8 , "clipped" 18 , or "pointed" 10 . The presence and morphology of this attribute can vary along a feather tract (e.g., Supplementary Fig. S6). Ontogenetic variation and sexual dimorphism of this attribute in coverts was also reported in Lucas & Stettenheim 1972 10 . Alular feathers can have angled distal tips as well 5,9 (not included, since they are not present in Archaeopteryx). An angled distal tip helps to increase aerodynamic lift by reducing tip vortices 18 . Table S1. Comparison of anatomical attributes shared by the isolated fossil feather and feather tracts of Archaeopteryx, based on the Altmühl*, Berlin 54 , and London specimens. Rows ranked according to Table 1. Consistency between the isolated feather and a given feather tract is designated "yes," "no," "maybe" (occasionally; superscript letters denote relevant specimens), or "?" (not observable) (see 15. Anatomical attributes). Superscript numbers denote additional references for individual attributes (column headings), as well as previous hypotheses of anatomical identity (boldface row headings). Abbreviations: UMPC, upper (dorsal) major primary covert; uMPC, under (ventral) major primary covert; UMSC, upper major secondary covert. *Note that elongate contours of the body and hindlimb 55 do exhibit lateral curvature (#5) and vane closure (#8), but are nonetheless inconsistent with the isolated feather with respect to all remaining observable attributes. , and Thermopolis (WDC-CSG-100) specimens. While such impressions are anatomically uninformative, those of the primary coverts on the Thermopolis specimen's left wing do suggest a posterior orientation (also described for the secondary coverts 29 ) consistent with that of the Altmühl and Berlin specimens' primary coverts (see main text).

Other
In the Maxberg specimen-the original of which has been lost since 1991-the wing feathers remained firmly attached 12,13 . We can infer that both surfaces of the wings are present, given that the left and right manus are in dorsal and palmar views on the main slab, respectively. On the counterslab, a furrow is visible on a partial rachis impression from a primary on the left wing, indicating the ventral surface ( Supplementary Fig. S13c). In the initial descriptions, Heller [30][31][32] identified numerous impressions covering the quills of the primaries that undoubtedly represent the primary covert feathers. However, the primary covert impressions we observed from photographs of nine casts of the main slab (n = 5) and counterslab (n = 4) were not distinct enough for assessing any anatomical attributes.   (1) 24.0° 27.8° (2) 26.5° 25.1° (3) (Fig. 4) 25.2° 23.1° (4) 24.

Designation.
Previous interpretations of the Munich slab (BSP 1869 VIII 1) as the main slab (e.g., de Beer 1954 16 ) may have been due to that half's accessioning prior to the Berlin slab (1869 vs. 1876 12,13 ), and/or the fact that von Meyer illustrated the Munich slab's outline and feather chirality (1862: Plate VIII, Fig. 3) 8 . However, von Meyer had access to both slabs 8 , and it has been hypothesized that he illustrated the fine details of the much better-preserved feather of the Berlin slab, by using a drawing mirror 38,17 . Given that this would yield a reflection that matches the feather on the Munich half, it makes sense that von Meyer would then draw or trace the outline of the Munich slab instead of the Berlin slab. Additionally, while von Meyer refers multiple times to the two halves as "both counterslabs" 14 2004 17 ), it may also reflect a natural differential in pigmentation. In the wing feathers of modern birds, the exposed surface is generally more melanized than the unexposed surface, for both visual and structural reasons 10,39 . If this were also the case in the fossil feather, the Berlin slab would thus represent the dorsal surface of the feather (more accurately, a mirror image imprint of it). This dorsal imprint interpretation is corroborated by microstructural evidence of the barbules, which Carney et al. 7 found to be "indistinguishable from those of modern pennaceous feathers with respect to morphology (Lucas & Stettenheim 1972) and barbule angles (Carlisle 1925)". In modern birds, feather barbicels always pass dorsally over the proximal barbules ( Supplementary Fig. S16f) 10,40 . In the Berlin slab, the fact that the preserved barbicels lie underneath the proximal barbule ( Supplementary Fig. S16c) reveals that we are observing the ventral surface of the feather microstructure (with the dorsal surface facing into the matrix). Thus, the darker and dorsal imprint of the Berlin slab, oriented by the narrower leading vane, indicates that the feather originated from the left wing of the animal. This left wing designation would likely hold true even if the feather were an under major or under median covert, as these "reversed coverts" have the same dorsoventral orientation as the upper coverts in modern birds (unlike the under minor coverts) 4,41-43 .

Melanosomes.
In Carney et al. 2012 7 , the presence of melanosomes in the isolated Archaeopteryx feather was supported by detection of elongate microbodies identical to melanosomes in their 1). size, 2). shape, 3). limited morphological range, 4). parallel alignment, 5). absence of serial arrangement (cf. bacterial chains), and 6). location (within barbules), as well as by 7). corroborating evidence of colour patterns from other fossil feathers 44 . This conclusion was criticized by Moyer et al. 45 , who recapitulated the obsolete interpretation 46 Fig. 1d-f 45 ), the extant melanosomes were inappropriately compared in cross-sectional view (Ibid. : Fig. 1b,c). The authors also state that in order to confirm the identity of microbodies in the fossil record, "molecular or chemical signals unique to either melanosomes or microbes in extant feathers" should be detected and "localized to the [fossilized microbody] structures to eliminate the alternative, using in situ surface techniques (e.g., time of flight secondary ion mass spectrometry (TOF-SIMS))". Yet, no such evidence was provided in support of their alternative microbe hypothesis. However, using TOF-SIMS, we subsequently detected 1). such molecular signals of melanin, 2). which were localized to melanosome-like microbodies 3). across a wide range of taxa, tissues, and environments-from the fossil feathers of the closely related Jurassic paravian Anchiornis 48 to the fossil skin of various marine reptiles 49 . Furthermore, 4). these molecular signatures were unique to animal eumelanin, and 5). no molecular signatures unique to microbes or microbial melanins were detected 48 . Together, this unequivocal and definitive molecular evidence of melanin and melanosomes in residues of fossil integument confirms our original interpretation, that the melanosome-like microbodies preserved in the isolated fossil feather are indeed melanosomes.

Colouration.
The distinct black and white reconstruction of the isolated fossil feather in Manning et al. 2013: Fig. 1F 50 (reproduced below) was based on the distributions of organic sulphur and trace metals, used as putative biomarkers for eumelanin. However, while we agree that sulphur can diagenetically bind to eumelanin, sulphur also binds to other organic matter (sulphurization) 7 . The latter is evident in the paper's own Fig. 1D 50 , where sulphur is bound to the plant matter surrounding the feather 18,12,13 . Similarly, the trace metals are abundant in the surrounding rock matrix of both the isolated feather (Manning et al. 2013: Fig. 1B,C 50 ), and especially the counterslab of the Berlin skeletal specimen (Ibid.: Fig. 3). Thus, the true plumage patterns of Archaeopteryx remain unknown. Fundamentally, sulphur and trace metals are indirect proxies that are not specific to eumelanin 51 , and therefore do not always indicate pigmentation patterns (false positive/type I error). By contrast, melanin residue and melanosomes are the actual pigment and pigment microstructures themselves, and therefore represent direct evidence of colouration. This direct evidence is also specific-here, the residue is constrained to the isolated feather structures and is not present in the surrounding plant matter or rock matrix 7 . Additionally, just because sulphur and trace metals are able to bind to eumelanin does not mean that they are always bound to eumelanin (false negative/type II error). Specifically, based on lower concentrations of copper, nickel, and organic sulphur (and speciously, a sulphate map), Manning et al. 50 reconstructed the feather's trailing vane as an "un-pigmented" white colour (below). However, white colour in feathers is produced by the absence of melanosomes 52 -therefore, if this area were truly white, there would be no melanosomes and certainly no melanin residue. Instead, this trailing vane is completely covered in dark melanin residue and contains thousands of observed melanosomes (e.g., Supplementary Fig. S16a-d). Thus, we reaffirm that the Archaeopteryx feather was entirely matte black, with a darker distal tip.