Molecular paleontology

How and Why I got involved in Molecular Paleontology- It was a natural from me - starting with coal chemistry - an ancient organic fossil! I just upped the analytical power with Soft X-rays! and great collaborators!

Background: I did my Ph.D. thesis at Penn State on the molecular structure of coal- and in order to do so I first had to acknowledge that coal was an organic fossil of primarily vascular plant debris- so in order to understand coal chemistry you had to start with plant chemistry.

What brought me into my research in "deep time" molecular paleontology was two fold:

1) During my Post Doc at Argonne National Laboratory I learned about the power of synchroton based Scanning Transmission X-ray Microscopy and micro-spectroscopy (on the C-, N- and O- K edges), and then

2) At GL I linked up with C. Kevin Boyce (now at Stanford). This was really a match made in science heaven. I pride myself as being a sold molecular spectroscopist , however I am not a paleontologist- I have no idea what to look for, which sample to look at, and why I would look at it- C. Kevin Boyce ( and later Derek Briggs, Dianne Edwards and John Nance) knew what to look at, what the questions were, and how my skills might aid the problem. I work for the paleontologists- I have the analytical skills and am willing and ready to collaborate!

So, once upon a time... Kevin Boyce (then a graduate student from Harvard) was wondering down the hall at GL with what I came to learn were "acetate peels" of ancient rocks. I asked Kevin what these were and when he explained to me what they were- I told him- "well I can use STXM to interrogate the carbon chemistry of the these ancient (carboniferous) fossils". The rest was history and continues to the present.

Figure 1: So on the left is an optical image of a Carboniferous Age acetate peel of a cross section of vascular plant fossil (a carboniferous tree fern). So ... I excised the region of interest with a scaple- mounted the little section in epoxy and then used the ultramicrotome to obtain ultra thin sections on the order of 140 nm thick! These I took to the X-ray synchrotron (in those days NSLS 1) and the first STXM (X1A) where we interrogated these samples using C- k edge spectroscopy and showed that yes even as this fossil was ~ 400 million years old - we could extract chemical information from it- You can see why a young (at that time) paleontologist like C. Kevin Boyce was excited! I was too!

Figure 2: So at this point, I was exploring the amazing ability to interrogate carbon chemistry at sub 100 nm resolution- Note: this was not possible until X1A (now everyone takes it for granted-yes this makes me sound like an old fart- but there you have it- in 1995 this was a revolution!!!). So in these early days I studied all sorts of things and here is really nice pair of images of microtomed Oak samples- BTW- I did and do my own microtoming. I do not have a lab manager.

In any event, the contrast in both images is based on chemistry- basically lignin and cellulose differentiation in the cell wall: I am still amazed when I see these images!

Well, Working with Kevin we found some really great applications of STXM in paleontology. As Kevin is a paleobotanist we focused on one of the outstanding problems- when did plants innovate lignin biosynthesis? To study this you need to understand the phylogeny of plants (Kevin does- I don't - or did'nt). I think that we likely did identify the key plant, Asteroxylon.

Searching for Evidence for the first biochemical innovation for the biosynthesis of lignin- a game changer for life and Earth! (A collaboration with C. Kevin Boyce, Sue Wirick and others).

Figure 3: So first Kevin and I went after an "easy" target, a classic chert locality (the Princeton Chert) that contained nicely fossilized samples of Metasequoia Milleri - a conifer- dated at 45 Myr. Kevin made an acetatate peel using HF to etch the chert and the standard acetone soaked cellulose acetate film for the peel. Once done, I excised a bit of longitudinal woody tissue, embedded that in epoxy and then ultra-microtomed that to provide ultra-thin sections on the order of 120 nm thick. These we took of to beamline X1A at the old NSLS synchrotron (where I learned about STXM!) and collected images like you see upper left. To obtain contrast that highlights the fossil you have to play some tricks- first of all you do not place the monochromator on a specific absorption band (as you are tempted to do) rather you go between strong absorptions (here we go to 286.3 eV). At this energy, only chemically complex things still absorb and bingo! We have a great image. You can still see that on the right side the embedding polymer is epoxy and on the left side the embedding polymer is cellulose acetate film. At the interface is our tiny little Metasequoia Milleri organic fossil, barely a single tracheid cell wide! The beauty is that you can see the chemical differentiation perfectly and using the micro-focusing capabilities- we can get very nice C-XANES spectra (on Right) - that clearly show the chemical differentiation that must be due to differential lignin/cellulose concentrations in the primary and secondary cell wall: As we see in modern plant vascular tissue (see above).

Figure 4: So then we went for the gold- gold standard that is for paleobotany- the Rhynie Chert from Aberdeenshire Scotland. This Devonian chert (~ 400 Myr old) contains some of the best preserved plant and insect fossils. Kevin was interested in a particular extinct plant - Asteroxylon - that lies very close to the phylogenetic root of all trees. Kevin painstakingly made an acetate peel of the Rhynie Chert- found a longitudinal fragment of Asteroxylon and I excised this, embeded this in epoxy and ultramicrotomed this. WOW! We found clearly indicated chemical differentiation. We interpret this signature as the signature of this plant having the capacity to biosynthesize both cellulose and lignin- if only cellulose- no chemical differentiation. We went on to go further down the "tree" of trees first to "Rhynia" and then to "Aglaophyton" - neither of which exhibited evidence of remnant chemical differentiation. This led us to propose that lignin biosynthesis emerged with Asteroxylon- and the rest was history- plants colonizing the continents, massive changes in continental weathering, the development of a floral canopy, changes in the Earth's albedo, carbon burial and the rise of O2.... yes you got it- we humans likely owe our existence to "little" asteroxylon learning how to biosynthesize lignin.

Perhaps the random mutation that led to the metabolic pathway to lignin- methoxy transferase - might have been as transformative to the Earth as Rubisco/photo synthesis in general. We owe our existence to plants!

P.S. My colleagues at Carnegie Plant Biology - Sue Rhee, Art Grossman and Devaki Bhaya have really opened my eyes to incredible biosynthetic landscape that are plant metabolic pathways. Starting with photosynthesis, to polycarbohydrate synthesis and construction, and then lignin biosynthesis- Plants have completely changed this world!!!!

What about other biopolymers- Exploring the ancient history of Chitin-Protein Complex in the Exoskeletons of really ancient arthropods- A collaboration with David Kilcoyne, Neal Gupta, Derrick Briggs and Roger Summons and others.

Figure XX: C- and N XANES imaging of modern Scorpion Cuticle: Each image is a "map" of chemistry. Note the scale bar is 5 microns. The image pixel size is 100 nm. Clearly resolved is a waxy outer region of the cuticle - likely to protect against loss of water. Note that peptide and protein/chitin complex are laminated- presumably for a good reason. In fact the degree of molecular differentiation in the modern scorpion cuticle is quite remarkable.

Figure 5 (LEFT) C-XANES of a pure Chitin standard (looks like it is- a polysaccharide); Modern Scorpion Exoskeleton- a nano composite of chitin and sclerotin protein- note that prominant amide peak!: the 310 Myr old scorpion cuticle and the 417 Myr old eurypterid cuticle- you see immediately the ancient fossils have taken a beating though diagnesis. you would be hard pressed to look at these C-XANES spectra and claim...."Ah Ha! Ancient cuticle here!" - that is the key- you need to have both the fossil context and the C-XANES to begin to understand diagenetic alteration of biomacromolecules- this is the beginning of deep time molecular paleontology IMHO!

FIgure 6: We determine atomic N/C and O/C from the atomic absorption cross sections for elements that are very well established. Above is an example fit for pure Chitin, but we can do this at 100 x 100 nm spot size- Thanks to STXM!

Back further to the Cambrian and really old and remarkably preserved Trilobites- Still working on these- have not yet published but could not resist showing these here! This is collaboration with David Kilcoyne, Yoko Kebukawa, Bob Hazen and me.

Figure 6: It appears that organic preservation is greatest for carbon within the carbonate matrix, where as the "free" organic carbon has taken a beating! Is it possible that some fraction of intact protein exists in this fossil? We don't know- for sure the carbon chemistry is more promising than I might have thought. We need to dig deeper here and we are!

Figure 7: N/C and O/C analysis reveals that the residual biopolymer in the Trilobite cuticle retains high nitrogen content consistent with presence of residual protein-chitin complex. High nitrogen content is definitely a biomolecular biomarker- That is we have no evidence that ancient kerogens can incorporate nitrogen- nor is there any evidence of such an event ever occurring. So when an ancient organic fossil has a lot of nitrogen, we have to believe that it is inherited.

Extremely well preserved polysaccharide and protein in 8-18 Ma Ecphora calcareous shell- Our work with John Nance (a collaboration with Marilyn Fogel, Bob Hazen, myself and John Nance).

Figure XX. Calvert Cliffs in Maryland that over look Chesapeake Bay and host mid-miocene fossli bearing strata. One of the more abundant fossils Ecphora, that is Maryland's state fossil. The Ecphora fossils are characterized with a redish brown exterior.

John Nance (of the Calvert Marine Museum and the University of Maryland) and came to the Geophysical laboratory with the question as to why Ecphora was the color it was. Marilyn and I recommended to John that he dissolve the carbonate shell to liberate the organics. The Ecphora shell dissolves easily in HCL and the residue was ribbon like organic with considerable flexibility and deep orange red color.

At GL we are very well equipped to to interrogate fossil organic solids. So I set out to apply 13C solid state NMR on the organic solids, Marilyn and I collaborated to do GC-MS analysis of aminoacids liberated from the hydrolysis of peptide and Marilyn performed carbon and nitrogen stable isotope analysis.

Figure XX: 13-C Solid State NMR spectrum of isolated Ecphora biopolymer. What we observe is remarkable preservation. ~ 70 % of the carbon exists as largely intact polysaccharide (note prominent secondary alcohols and the glycosidic carbon ~ 103 ppm- the linkage between sugars in intact polysaccharide - and the weakest bond! next to peptide) and intact peptide (~ 175 ppm). But ~ 30 % of the carbon is disordered aromatic/olefinic carbon (~ 110 to 160 ppm) that we interpret as diagnetic alteration products of sugars and amino acids, classically known as Mallaird products.

A large fraction of the carbon appears to be nearly pristine biomacromolecule- Protein and polysaccharide.

Ready for more fossils- contact me! Cheers!

Figure XXX: Marilyn Fogel took the organic solids (biopolymer) isolated by HCL digestion and subjected this to standard protein hydrolysis conditions to liberate individual amino acids. These were then derivatized first with 2-propanol and acetyl-chloride and then trifluoroacetic acid anhydride. These derivitzed amino acids were now ready for analysis via GC-MS as a solution in dichloromethane. The total ion chromatogram is shown were many amino acids are resolved along with a large amount of unidentified organics that are likely products of organic diagenesis.

The detected amino acids likely are only observable due to the hydrolysis, suggesting intact peptide - as is strongly suggested in the 13C NMR spectrum above.

There is more to come here- please stay tuned! If you are interested in collaborating- well please contact me. HONK if you support STXM and NMR! and Isotopes!!!!