Literature Examples Highlighting the Potential of Molecular Histology as a Proxy
A few previous studies have examined fossil/sub-fossil molecular histology in a manner that can be linked to preservation potential for molecular sequences. A discussion of some findings relevant to the correlation of molecular histology with degree of sequence preservation follows.
A 2007 study by Schweitzer et al. used light and electron microscopy to survey the molecular histology of bone specimens ranging from modern day through to the Triassic33. The study reported that the molecular histology (especially the “collagenous” matrix) of specimens with dates exceeding 100-300Ka was substantially altered relative to specimens of younger timepoints. Light microscopy was herein used to replicate and reevaluate reported data for three of the 2007 study specimens (data not shown), the M. columbi femur (MOR 501 (formerly MOR 91.72), ~12Ka), the M. columbiskull fragment (MOR 604, ~100-300Ka), and the M. americanum rib (MOR 605, ~100-300Ka). This was done according to the same demineralization protocol reported by the 2007 study33.
Collagenous matrix of the mid-Pleistocene MOR 604 and MOR 605 specimens was highly fragmented and brittle, supporting substantial degradation. Histological structures resembling blood vessels readily broke free of the degraded matrix and were easily isolated. Both specimens exhibited evidence of exogenous, orange-brown mineralization across portions of structure surfaces even after hydroxyapatite was removed via acid demineralization. In contrast, the late Pleistocene MOR 501, also from the temperate region of Montana, U.S.A.33, preserved a structured, relatively intact collagenous matrix. No evidence for exogenous mineralization of MOR 501 was detected with light microscopy.
Matrix from MOR 501 was more consistent in morphology with past reports on extant collagenous matrix32, 41 than what was observed for matrix of MOR 604 and MOR 605. Collagenous matrix of MOR 604 and MOR 605 was closer in morphology to what has previously been reported for Mesozoic dinosaurs32-34 and early-mid Cenozoic organisms38, 39. The stark difference in these observations supports a disparity in degree of type-1 collagen preservation between these specimens, which is predicted to affect potential sequencing analyses. Prior studies have reported type-1 collagen sequences from MOR 60462 and MOR 60557. MOR 501 however has not previously been sequenced and a direct comparison regarding degree of type-1 collagen sequence preservation is not currently possible. Further, MOR 501, MOR 604, and MOR 605 were all recovered from the same geographic region, albeit different burial sites33. This supports the observed dichotomy in “collagenous” matrix preservation is thus likely less dependent on thermal setting.
Another study that analyzed molecular histology to a limited extent is that of the Pliocene Ellesmere Island camel tibia2. A cross-section of a vascular canal within the tibia was elementally mapped using energy dispersive X-ray spectroscopy (EDS). The analysis demonstrated that elements consistent with iron oxyhydroxides and barium sulfates co-localized to the vascular canal. The presence of such exogenous minerals supports that this tibia had undergone substantial chemical alteration. Both mineral precipitants are consistent with observations from older tertiary37-39 and even Mesozoic specimens16, 35, 36, 40, 41, and certainly precludes it from being considered a “sub-fossil”. Despite the apparent chemical alteration to its molecular histology, the tibia still preserved collagen sequences identifiable via mass spectrometry2.
Samples from the Pliocene tibia were not demineralized and examined with light microscopy within the study2, however, thus precluding a direct comparison against observations for the MOR 501, MOR 604, and MOR 605 “collagenous” matrix morphology. The substantial mineralization detected by the EDS analysis is consistent with observations of mineralized histological structures within MOR 604 and MOR 60533. This supports a hypothesis that any “collagenous” matrix the Ellesmere Island tibia preserves is likely highly degraded morphologically, in a manner consistent with MOR 604 and MOR 60533 as well as previous reports for Mesozoic dinosaurs33 and pre-Pliocene Cenozoic37-39 specimens.
Data from the two studies above already enables some predictions to be made regarding the relationship of the specimens’ underlying molecular histology with degree of sequence preservation, and even some diagenetic variables. The dichotomy in molecular histology between extant specimens along with MOR 501 when compared against MOR 604, MOR 605, the Pliocene camel tibia, and Mesozoic dinosaurs is to this point a largely unexplored finding. Few, if any, studies have directly explored how these differences in “collagenous” matrix histology manifest in degree of recovered sequence data.
Based on the discussion above, bone specimens preserving ancient DNA are herein hypothesized to possess an intact, relatively robust collagenous matrix like that of MOR 501. If sequence-able DNA is present, collagenous matrix would also still be expected to be relatively intact. In contrast, bone specimens with a brittle, easily fragmented “collagenous” matrix like that of MOR 604 and MOR 605 are predicted to preserve, at most, remnant peptide sequences. If the collagenous matrix has degraded to the point it has lost structural integrity, the preservation of sequence-able DNA is not expected18, 27, 59. Further, this agrees with the trend of sequence-able DNA being rarely reported from specimens exceeding 0.13-0.24Ma in geologic age (excluding cave and permafrost deposits) as MOR 604 and MOR 605 are both assigned dates of ~100-300Ka33.
If such a hypothesis were supported, practical methods such as electron and even light microscopy may be capable of screening fossil/sub-fossil specimens for sequence preservation. However, the limited extent of the data that has been reported for ancient vertebrate molecular histology severely limits the conclusions that can be drawn regarding these relationships. This epitomizes the need emphasized by this review for extensive study of fossil vertebrate molecular histology.