Localization of antibody antigen (ab—ag) complexes <i>in situ</i> on feathers exposed to varied conditions.

<p>(A-B) Control, (C-D) 60°C, and (E-F) 350°C feathers. White barbs (A and C) are compared with brown barbs (B and D) for both the control and 60°C conditions. There are no noticeable differences in strength of binding between the pigmented and non-pigmented barbs in either condition. Antibodies bind with greater avidity in the feathers treated at 60°C, consistent with what we have observed in samples from other experiments that are partially degraded. (E) Background signal in the 350°C condition is weak and diffuse but binding is greater than the secondary antiserum only negative control (F) and localized to the keratinous ‘struts’ within the pith.</p

Biofilm growing on cow bone from which organics had been removed (see methods), 48 hours after inoculation.

<p>(A) <i>B</i>.<i>cereus</i> has colonized the bone and can be seen growing on and around the bone at low magnification. (B) <i>S</i>.<i>epidermidis</i> at higher magnification. Note the interaction of the biofilm with the surface of the bone (yellow arrows). Similar observations were made for both organisms. (C) Feather (tan) and ostrich eggshell (white) in the presence of <i>B</i>. <i>cereus</i> shows biofilm growth on tissues and in surrounding medium. (D) Feather and eggshell surrounded by biofilm from <i>S</i>. <i>epidermidis</i>, as above.</p

Feathers after 10 year exposure to several different environmental conditions.

<p>(A-B) Room temperature control feathers. Pigmented (reddish-brown) and non-pigmented (white to beige) patterning is visible with no signs of degradation. (C-D) Wet burial (60°C) feathers show signs of degradation and color change. Bands of pigmentation are still visible (arrows), although the white-beige parts appear more yellow and the patterning observed in control feathers are obscured. (E-F) 350°C feather pieces appear as small shiny black fragments which are associated with reddish-brown sediment. The pieces are not able to be identified to specific parts of the feather.</p

Supplemental Methods and Figures from Microscopic and immunohistochemical analyses of the claw of the nesting dinosaur, <i>Citipati osmolskae</i>

Supplemental Methods and Figures for Moyer et al 201

Localization of antibody antigen (ab—ag) complexes <i>in situ</i> on <i>Shuvuuia deserti</i> (IGM 100/977) filament.

<p>(A) Shows positive binding of the anti-chicken feather antiserum to the fossil tissue as indicated by the green fluorescent signal. (B) Demonstrates that the binding in A) is localized specifically to the tissue. (C and D) Secondary antiserum only control is negative and shows that the positive signal in A) is not due to spurious antibody binding.</p

TEM images of 350°C feather fragments.

<p>(A-B) Unidentifiable piece of 350°C feather at lower and higher magnification, respectively. The honey-comb structure observed in (A) indicates it is the pith of either a barb or rachis. (C-D) Feather fragment positively identified as barb. (C) External cortex (arrow) and internal pith are observed. (D) At higher magnification no electron dense microbodies consistent with melanosomes are observed in the cortex.</p

Barb fragment from 350°C feather.

<p>(A) Barb ramus retaining barbule protrusions (arrows) allows definitive identification as a barb fragment. Only the most proximal parts of the barbules can be observed where they branch from the ramus. (B) Transmitted light image of a 200nm thin section of a barb from the 350°C treated feather (similar to A) shows presence of external cortex (arrowhead) and inner pith observed as honey-comb texture.</p

TEM images of feathers from varied conditions.

<p>(A-B) Represent unpigmented barbs from the control feather and 60°C feather respectively. (C- D) Brown barb and barbule from control feather. (C) Represents barb where cortex (arrow) and inner pith are visible. Note melanosomes (arrowhead) are sparse but present only in barb cortex. (D) Barbule with melanosomes (arrow) from control feather. (E) 60°C brown barb with barbule extending from the left side. Melanosomes are concentrated in the barbule and appear partially degraded as indicated by the less dense (arrowhead) and even ‘hollow’ (arrow) centers observed in the inset. (F) Pith from a rachis taken from the 60°C condition, embedded and sectioned separately (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157699#sec005" target="_blank">Methods</a>). Note: Whether pith derives from the rachis (F) or barb (C) is impossible to determine at this level of magnification.</p

Expansion for the <i>Brachylophosaurus canadensis</i> Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a <i>Brachylophosaurus canadensis</i> specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, <i>B. canadensis</i> is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, <i>B. canadensis</i> is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of <i>B. canadensis</i> collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087

Expansion for the <i>Brachylophosaurus canadensis</i> Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a <i>Brachylophosaurus canadensis</i> specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, <i>B. canadensis</i> is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, <i>B. canadensis</i> is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of <i>B. canadensis</i> collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087