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.