Review of Palaeobotany and Palynology 233 (2016) 169–175

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Review of Palaeobotany and Palynology

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In situ confocal laser scanning microscopy and Raman spectroscopy ofbisaccate pollen from the Irati Subgroup (Permian, Paraná Basin, Brazil):Comparison with acid-macerated specimens

J.William Schopf a,b,c,d,⁎, Cléber Pereira Calça e, Amanda K. Garcia a,b, Anatoliy B. Kudryavtsev b,d, Paulo A. Souza f,Cristina M. Félix f, Thomas R. Fairchild e

a Department of Earth, Planetary, and Space Sciences, University of California, 595 Charles E. Young Drive East, Los Angeles, CA 90095, USAb Center for the Study of Evolution and the Origin of Life, University of California, 595 Charles E. Young Drive East, Los Angeles, CA 90095, USAc Molecular Biology Institute, University of California, 495 Hilgard Avenue, Los Angeles, CA 90095, USAd Department of Geosciences, University of Wisconsin Astrobiology Research Consortium, University of Wisconsin, 1215 W. Dayton St., Madison, WI 53706, USAe Instituto de Geociências, Universidade de São Paulo, Rua do Lago 562, São Paulo, SP CEP 05.508-080, Brazilf Instituto de Geociências, Universidade Federal do Rio Grande do Sul, Bento Gonçalves 9500, Porto Alegre, RS CEP 91.540-000, Brazil

⁎ Corresponding author at: Department of Earth, PUniversity of California, 595 Charles E. Young Drive EasTel.: +1 310 825 1170.

E-mail address: schopf@epss.ucla.edu (J.W. Schopf).

http://dx.doi.org/10.1016/j.revpalbo.2016.03.0040034-6667/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 October 2015Accepted 26 March 2016Available online 19 April 2016

Acid maceration, used to isolate compression-preserved flattened spores and pollen from fine-grained clasticrocks, can yield copious quantities of palynomorphs and high-quality morphological information. Such macera-tion, however, is generally not applicable to organic-walled microfossils three-dimensionally permineralized inchemically precipitated rocks (e.g., most fossiliferous cherts), its use resulting in disintegration or destructionof the fossils as they are freed from their embedding supporting matrix. In this study of bisaccate pollen grainspermineralized in stromatolitic chert of the late Early Permian Assistência Formation (Irati Subgroup) of south-eastern Brazil, we compare the morphology of specimens imaged by scanning electron microscopy in acid-resistant residues with that of grains embedded in petrographic thin sections and imaged by transmitted lightoptical microscopy, confocal laser scanning microscopy, and Raman spectroscopy. The results document numer-ous benefits of the use of these three techniques for studies of permineralized palynomorphs in situ.

© 2016 Elsevier B.V. All rights reserved.

Keywords:Acid macerationPermineralizationPetrographic thin sectionRaman Index of PreservationThree-dimensional imagery

1. Introduction

Although the most “lifelike,” best preserved fossils in the geologicalrecord are three-dimensionally permineralized (“petrified”) in quartz,calcite, apatite, or gypsum, the vast majority (N99%) of describedPhanerozoic organic-walledmicrofossils (e.g., pollen, spores, acritarchs,and microalgae) are preserved as compressions, two-dimensional flat-tened remnants compressed within siltstones or shales. Thus, andthough studies of permineralized fossils in situ can be appreciablymore morphologically informative than those of flattened specimens,compression-preserved palynomorphs, bothwidespread and especiallywell known, have been studied intensively by palynologists.

To identify such specimens and document their morphology,most workers dissolve the microfossil-hosting rocks in mineral acids(e.g., HF, HCl) and study the macerated acid-resistant organic residues

lanetary, and Space Sciences,t, Los Angeles, CA 90095, USA.

by use of optical and scanning electron microscopy (“SEM”). However,SEM cannot be applied to studies of rock-encased (rather thansurface-exposed) permineralized fossils, and because the cell walls ofpermineralized fossils are mineral-infused and supported by their em-bedding matrix, acid maceration commonly results in their disaggrega-tion or destruction.

A solution to this problem is documented here by comparison ofresults obtained from studies of quartz-permineralized bisaccatepollen grains imaged by SEM in acid macerates of a richly fossiliferouscherty stromatolitic unit (Calça and Fairchild, 2012) of the late EarlyPermian Assistência Formation (Irati Subgroup) of southeastern Brazil(Hachiro, 1996) with those of rock-embedded specimens imaged bytwo techniques recently introduced to such studies, confocal laser scan-ning microscopy (“CLSM”) and Raman spectroscopy. Freed from theirencasing rock matrix, the acid-macerated palynomorphs provide rela-tively limited usefulmorphological information. In contrast, comparablestudies in situ by use of CLSM (which images their morphology in threedimensions) and Raman spectroscopy (which images their carbona-ceous composition and documents their fidelity of preservation) yielduseful data at submicron spatial resolution. Because both of these

Fig. 1. (A) Raman spectrum of kerogen that comprises the body wall of a bisaccate pollen grain that transects the upper surface of a thin section (Plate II, 4 through II, 12) showing its “D”and “G” Bands and the strong laser-induced background fluorescence of this geochemically immature carbonaceous matter that obscures the much weaker kerogen Raman-signal. (B)Kerogen Raman spectrum of this Permian (~278 Ma) Assistência Fm. bisaccate grain, with the fluorescence background subtracted, compared with those of similarly quartz-permineralized kerogenous microfossils in stromatolites of the Bitter Springs Fm. (~800 Ma), Gunflint Fm. (~1900 Ma), Allamoore Fm. (~1060 Ma), Skillogalee Dolomite (~760 Ma),Auburn Dolomite (~720 Ma) and the River Wakefield Fm. (~725 Ma), ordered from top to bottom by the increasing geochemical maturity of their kerogenous components, quantifiedby the Raman Index of Preservation, “RIP” (Schopf et al., 2005).

Fig. 2. Stratigraphic setting of the stromatolitic fossiliferous cherts studied here from theEvaporite Bed of the Assistência Formation (Morro do Alto Member, Irati Subgroup,Passa Dois Group), following the nomenclature of Hachiro (1996).

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techniques are non-destructive, they can be used to analyze especiallyimportant specimens such as those archived in museum collections.

2. Fossil preservation and analytical techniques

2.1. Permineralization- vs. compression-preservation

The Assistência Formation gymnospermous bisaccate pollen studiedhere is preserved by permineralization (cf. petrified wood), the mostlifelike mode of preservation known (Schopf, 1975; Schopf andKudryavtsev, 2010). Their fossil-enclosing matrix, a chemically precipi-tated chert, is composed of an interlocking three-dimensional mosaic ofpolyhedral cryptocrystalline quartz that, deposited from colloidal solu-tion, infused cell walls and permeated cell lumina. The silica later trans-formed, with loss of water, through an opaline state and subsequentlycrystallized to form the mineralic mosaic. In essence, the quartz has re-placed cellular water – including the intra- and inter-micellar water ofthe linear organic polymers of the cell walls as well the aqueous cyto-plasm (Schopf, 1975) – and the crystallized quartz grains transect, butdo not disrupt, the morphology of the three-dimensionally preservedpollen. Permineralization is thus analogous to the embedding of cellsin epoxy resin for transmission electronmicroscopy, the permineralizedfossils however being a mixture of infused quartz and carbonaceousorganic matter (now kerogen) rather than of resin and originalbiomolecules.

The fidelity of preservation by permineralization, not alwaysyielding excellently preserved fossils, varies with the robustness andthickness of the walls of the permineralized cells (those of vascularplants more commonly being better preserved than thinner-walledpalynomorphs and microscopic fungi and prokaryotes); the time ofmineral-infusion (whether early or late during diagenesis); the grainsize and related characteristics of the permineralizing mineral(e.g., whether cryptocrystalline and three-dimensionally interlocking,like those of cherts, or larger, lath-shaped, and cell lumina-infilling,like those typical of calcitic coal balls); and the post-depositional ther-mal alteration of the deposit (e.g., Schopf, 1975; Wellman et al., 2006;Stevens et al., 2010).

In contrast with the three-dimensional cellular preservation provid-ed by permineralization, compression-preserved fossils, those mostcommonly studied by palynologists, are typically flat, compressed be-tween layers of fine-grained clastic sediments. Such preservation by

compression distorts original morphology and can affect taxonomicallypotentially useful characters.

2.2. CLSM and Raman spectroscopy

Introduced to paleobiologic studies of permineralized fossils duringthe past decade (Schopf et al., 2006), confocal laser scanning microsco-py is capable of providing both two- and three-dimensional images oforganic-walled fossils at ~0.2 μm lateral spatial resolution (e.g., Schopfand Kudryavtsev, 2009, 2010). To do so, CLSM detects laser-inducedfluorescence emanating from the interlinkedpolycyclic aromatic hydro-carbons that comprise kerogen, the carbonaceousmatter of such fossils.CLSM can thus provide confirming evidence of the kerogenous nature ofeven veryminute fossils, themolecular–chemical composition of whichcan be established by use of Raman spectroscopy. In addition – and ofparticular usefulness in palynology – the resulting images, if processedlike those shown here, can be rotated to illustrate a specimen excellent-ly from multiple perspectives (Supplementary video).

Fig. 3.Map showing the distribution of Carboniferous and Permian sedimentary rocks in the Paraná Basin of southeastern Brazil and the location of the Paraisolândia District (CharqueadaMu-nicipality) fromwhich the studied fossiliferous chertswere collected fromoutcrop;MG:Minas Gerais;MS:Mato Grosso do Sul; PR: Paraná; RJ: Rio de Janeiro; SC: Santa Catarina; SP: São Paulo.

Fig. 4. (A) Silicified dolomite of the Permian Assistência Formation in the ParaisolândiaDistrict at the locality from which were collected the stromatolitic fossiliferous chertsstudied here (denoted by the white rectangle). (B) Stratiform stromatolite containingthe laterally continuous thin black (carbonaceous) chert laminae that host the pollenstudied here and diverse other cellularly permineralized fossils (Calça and Fairchild,2012).

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Although CLSM has been used previously to image organic-walleddinoflagellate cysts (e.g., Feist-Burkhardt and Pross, 1999) and com-pressed gymnospermous and angiosperm-like pollen (Hochuli andFeist-Burkhardt, 2004, 2013) isolated by acidmaceration fromMesozoicfine-grained clastic sediments, the present study of the Assistência For-mation bisaccate pollen is evidently its first application in situ to pollengrains three-dimensionally preserved by permineralization in chemi-cally precipitated rocks.

Raman spectroscopy, now an established technique in paleobiologicstudies, was first applied to permineralized fossils some 15 years ago(Kudryavtsev et al., 2001; Schopf et al., 2002). Like CLSM, confocalRaman-based molecular–chemical mapping – having a lateral spatialresolution of ~0.8 μm – can provide both two- and three-dimensionalimages (e.g., Schopf and Kudryavtsev, 2009, 2010, 2012). In contrastwith CLSM, however, Raman can identify definitively the carbonaceouskerogen of organic-walled fossils (documented by its principal “D” and“G” Raman spectral bands) as well as the mineralogy of the associatedmatrix. In addition, by use of a simple metric, the Raman Index of Pres-ervation, “RIP” (Schopf et al., 2005, pp. 359–361), Raman spectra can beused to assess the geochemical maturity of the kerogen comprisingpermineralized fossils, an indicator of their fidelity of preservation.

Although the geochemically relatively immature kerogen compris-ing the pollen studied here produces laser-induced Raman-signal-ob-scuring strong fluorescence (Fig. 1A), subtraction of this fluorescencefrom the spectrum yields an RIP of 9.1. Thus, this Permian Assistênciaorganic matter is better preserved (less geochemically altered) thanthat comprising fossils of the Precambrian Bitter Springs (RIP = 9.0),Gunflint (RIP = 8.8), and many other stromatolitic cherts (Fig. 1B;Schopf et al., 2005, Fig. 9).

3. Geologic setting, materials, and methods

3.1. Geologic setting

The fossiliferous chert studied is from a stromatolitic unit of theEvaporite Bed in the lower part of the ~278 Ma late–Early PermianAssistência Formation of the Irati Subgroup, Passa Dois Group (Fig. 2),situated in the Paraisolândia District (Charqueada Municipality) ofthe state of São Paulo, southeastern Brazil (Fig. 3), where it wascollected from outcrop (Fig. 4). Fossil palynomorphs (e.g., Souza andMarques-Toigo, 2003, 2005; Santos et al., 2006; Premaor et al., 2006),plants, and invertebrates are abundant in the Irati Subgroup (Holz

et al., 2010), but it is especially well known for its preserved Gondwanamesosaurid reptiles (e.g., Oelofsen and Araújo, 1983; Chahud and Petri,2013). Taxonomic analyses of the bisaccate palynomorphs illustratedhere will be presented in a subsequent contribution that will addressall components of the diverse permineralized assemblage of theAssistência Formation cherts.

3.2. Acid-macerates and thin sections

At the Systematic Paleontology Laboratory of the Geoscience Insti-tute, University of São Paulo, palynological acid macerations were pre-pared using 40% HF, 10% HCl, and decantation and pipetting (ratherconventional centrifugation and sieving). Using standard techniques,petrographic thin sections were prepared of parts of the unprocessed

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rock samples (University of São Paulo Palynological Collection CodesGP/4E 1492–1706).

3.3. Optical, scanning electron, and confocal laser scanning microscopy

Optical images of thin section-embedded specimens were acquiredat the University of California, Los Angeles (UCLA) using fluorescence-free microscopy immersion oil and a Leitz Orthoplan 2 microscopeequipped with a Nikon DS Microscope Digital Camera. The SEM imagein Plate I, 2 was acquired of a Fe-coated specimen by use of a TescanVega TC scanning electron microscope at the University of Johannes-burg, South Africa. All other SEM images were obtained of Au- or C-coated specimens using a high-resolution Oxford SEM instrument atthe University of São Paulo.

Confocal laser scanning micrographs were obtained at UCLA usinganOlympus Fluoview300 confocal laser scanning biologicalmicroscopesystem equippedwith twoMelles Griot lasers, a 488 nm20mW-outputargon ion laser, and a 633 nm10mW-output helium-neon laser. Imageswere acquired using a 100× oil-immersion objective, fluorescence-freemicroscopy immersion oil, and filters in the light-path to removewave-lengths b510 nm (for 488 nm laser excitation) and b660 nm (for633 nm laser excitation) from the laser-induced fluorescence emittedby the specimens. Image-sets were subsequently processed by use ofthe VolView v3.4 3D-rendering computer program that permits imagemanipulation in three dimensions.

3.4. Raman spectroscopy

Molecular–structural compositional analyses of the fossils and associ-ated minerals were carried out at UCLA using a T64000 triple-stage con-focal laser-Raman system that permits acquisition both of point spectraand of Raman images that display the two-dimensional spatial distribu-tion of the molecular–structural components of the specimens and theirassociated minerals. A Coherent Innova argon ion laser provided excita-tion at 457.9 nm permitting data to be obtained over a range from ~300to ~3000 cm−1 using a single spectral window centered at 1800 cm−1.The laser power used was ~6–8 mW over a ~1 μm spot, a configurationwell below the threshold resulting in radiation damage to kerogenousfossils, and the thin sections were covered by a veneer of fluorescence-free microscopy immersion oil, the presence of which has been shownto have no discernable effect on the Raman spectra acquired (Schopfet al., 2005). Varying pixel intensities in the two-dimensional Raman im-ages, acquired at the ~1600 cm−1 “G” band of kerogen, correspond to therelative concentrations of carbonaceous matter.

Plate I. Comparison of chert-permineralized bisaccate gymnosperm pollen grains from the Iratisolated in acid-resistant macerates (1–3) with grains analyzed in situ, in petrographic thin se(4, 7, 10, 13), two-dimensional confocal laser scanning microscopy, “CLSM” (5, 8, 11, 14), and

(1-3) Scanning electron micrographs of representative bisaccate grains (coated respectively(4-6) A bisaccate grain in situ, oriented parallel to the thin section surface, showing in comp

and smooth surface of the pollen body illustrated in the 2-D CLSM image (5, face view)petrographic thin section (“P.T.S.”) PAR-14; microscope stage coordinates (“S.C.”) 45.

(7-9) A series of images comparable to (4–6) of a grain in which the proximal part of the ceE.F.S. R45[0].

(10-12) Images comparable to the foregoing of a grain oriented tangential to the thin section susurface of the section; P.T.S. PAR-14; S.C. 8.2 × 98.6; E.F.S. T7[0].

(13-15) A series of images comparable to the foregoing showing a grain overlain by an ellipsoisurface of the thin section; P.T.S. PAR-14; S.C. 32 × 102.4; E.F.S. Q31[0].

Plate II. Chert-permineralized bisaccate gymnospermpollen grains from the Irati Subgroup (Perthin sections imaged by optical microscopy (1, 4, 7, 10), two-dimensional confocal laser scanninfor which images were acquired at ~1603 cm−1, the “G” band of kerogen that documents the

(1, 2, 3) Three images that showwhat appears to be two juxtaposed grains, a saccus of one grai14; microscope stage coordinates (“S.C.”) 22.4 × 102; England Finder slide coordinate

(4, 5, 6) A grain oriented parallel to the upper surface of the thin section that transects the polleshow a part of the proximal wall of the central body (7–9) and a part of the outer wa

(7) Optical, (8) CLSM, and (9) Raman images of the area denoted in (4) showing the exin(10) Optical, (11) CLSM, and (12) Raman images of the area denoted in (4) showing the s

4. Results and discussion

SEM images of bisaccate pollen grains in acid-resistant residues ofthe Assistência stromatolitic chert are shown in Plate I, 1 through I, 3for comparison with similarly bisaccate grains chert-embedded and an-alyzed in situ by use of opticalmicroscopy and CLSM(Plate I, 4 through I,15) and of optical microscopy, CLSM, and Raman spectroscopy (Plate II,1 through II, 12).

4.1. Benefits of optical, CLSM, and Raman studies in situ

Optical microscopy and SEM of acid macerates initially preparedusing conventional centrifugation and sieving of the acid-resistant resi-due yielded only fragmented, degraded, and partially or completelydestroyed specimens. Modification of the standard processing proce-dure, by using decantation and pipetting rather than centrifugationand sieving, yielded the grains shown in Plate I, 1 through I, 3, the bestpreserved such specimens detected. SEM images of these maceratedspecimens provide useful morphological information, showing identifi-able sacci, some exhibiting discernable alveolar structure. These imagesalso show that the central bodies of the grains have only partially sur-vived maceration and that their SEM imagery provides limited taxo-nomically diagnostic information. Such marginally useful results aretypical of studies of acid-macerated permineralized microfossils that,unlike studies in situ, commonly yield only bits and pieces of degradedand disintegrated fossil specimens.

In contrast with studies of such macerates, CLSM imaging of therock-embedded grains in situ provides high-quality information aboutthemorphology of their sacci and central bodies. Compare, for example,the SEM image in Plate I, 1, which shows the alveolar ornamentation ofone of the sacci of this acid-macerated specimen, with the morphologi-cally more informative CLSM images of intact chert-embedded speci-mens (e.g., Plate I, 5, I, 8, I, 11, I, 14 and Pl II, 2, II, 5). Although thecentral bodies of themacerated grains are partially or largely degraded,the CLSM images of the rock-encased grains show them to be intact, andfor a surface-exposed specimen CLSM documents the sub-structure ofits walls (Plate II, 8, II, 11). Moreover, the CLSM-detectable fluorescenceof the in situ specimens reveals detailed morphological informationeven of grains that in optical images appear to be poorly and incom-pletely preserved (compare Plate I, 4 with I, 5 and Plate I, 7 with I, 8).And, unlike SEM or optical microscopy which yield face-views only,CLSM acquires images in three dimensions (Plate I, 6, I, 9, I, 12, I, 15)that provide morphological, biological, and taxonomic information at

i Subgroup (Permian, Paraná Basin, Charqueada Municipality of the Paraisolândia District)ctions (4–15), imaged by scanning electron microscopy, “SEM” (1–3), optical microscopythree-dimensional CLSM (6, 9, 12, 15).

with Au, Fe, and C) showing the variably degraded morphology of macerated specimens.arison with the optical image (4) the better resolution of the alveolar structure of the sacciand the three-dimensionality of the specimen evident in the rotated 3-D CLSM image (6);5 × 100.8; England Finder slide coordinates (“E.F.S.”) R45[4].ntral body transects the upper surface of the thin section; P.T.S. PAR-14; S.C. 44.8 × 101.1;

rface such that a part of one saccus, exhibiting alveolar ornamentation, transects the upper

dal body (possibly a unicell or a part of the saccus of second grain) that transects the upper

mian, Paraná Basin, CharqueadaMunicipality of the Paraisolândia District) in petrographicgmicroscopy, “CLSM” (2, 5, 8, 11), and two-dimensional Raman spectroscopy (3, 6, 9, 12)distribution of carbonaceous matter (blue). (see on page 174)

n transecting the upper surface of the thin section; petrographic thin section (“P.T.S.”) PAR-s (“E.F.S.”) Q22[0].n body and both sacci; the black rectangles in (4) denote areas that at highermagnificationll of a saccus (10–12); P.T.S. PAR-14; S.C. 14.1 × 105.2; E.F.S. M13[4].e of a part of the proximal body wall.tructure of a part of the outer wall of a saccus.

Plate I.

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high (~0.2 μm) spatial resolution and that can be rotated and viewedfrom multiple perspectives (Supplementary video).

Use of Raman spectroscopy in such studies (applicable also tocompression-preserved palynomorphs) yields additional benefits.

Individual fossil specimens can be analyzed by Raman as well as by op-tical microscopy and CLSM – each of which is non-destructive and non-intrusive – to provide mutually reinforcing information (Plate II, 1though II, 12).Moreover, Ramandocuments definitively the kerogenous

Plate II. (caption on page 172).

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composition of fossils (Fig. 1), whereas the carbonaceous content of fos-silized materials can only be inferred from CLSM-detected fluorescence(which can be produced by sources other than kerogen) or from theamber to dark brown color of fossils (that can bemimicked by hematiteand various opaque minerals).

In addition, as noted above (CLSMand Raman spectroscopy), by use ofthe Raman Index of Preservation, the “RIP,” Raman spectra can be usedto assess quantitatively the geochemical maturity of the kerogenouscomponents of permineralized organic-walled fossils, a more objectiveanalytical measure of their fidelity of preservation than the color-

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based Thermal Alteration Index, the “TAI” (Staplin, 1982), commonlyused by palynologists for specimens in acid-macerates. And the RIP,which requires only themeasurement of peak heights of Raman spectra(Schopf et al., 2005), is more easily applicable to such analyses than therecently proposed Palynomorph Darkness Index, the “PDI” (Goodhueand Clayton, 2010) – a proxy for measurement of vitrinite reflectancethat requires color calibration among the specimens analyzed. In con-trast with the RIP, by which diverse permineralized microfossils havebeen analyzed in more than 30 geological units (e.g., Schopf et al.,2005; Schopf and Kudryavtsev, 2009, 2012), neither the TAI nor thePDI has evidently been applied to permineralized organic-walled fossils.

Finally, the combined use of optical, CLSM, and Raman imagery forstudies of palynomorphs in petrographic thin sections can be used to doc-ument their preservational context, the spatial relations of the fossils tosedimentological features and to other members of a preserved commu-nity, a component important to their paleoenvironmental and taphonom-ic interpretation that cannot be provided by studies of acid macerates.

4.2. Limitations of these techniques

Neither CLSM nor Raman, techniques relatively new to studies ofpermineralized fossils, is a panacea. Although CLSM provides lateralspatial resolution of ~0.2 μm – more than 30% greater than the~0.3 μm resolution afforded by optical microscopy – the resolution itprovides is much less than that available from use of SEM. And likeSEM, neither CLSM nor Raman documents the true color of the speci-mens analyzed, used by palynologists to infer carbonaceous composi-tion and fidelity of preservation. In addition, it should be noted thatthe taxonomy of fossil palynomorphs is currently based almost entirelyonflattened compression-preserved specimens rather than on themorelifelike three-dimensionalmorphology exhibited by permineralized fos-sils such as those studied here.

5. Conclusion

The goal of this study is to present a solution to the problem posed topalynology by permineralized rock-embedded palynomorphs forwhichacid maceration typically yields marginal results. The results presented,based on the combined use of optical microscopy, confocal laser scan-ning microscopy, and Raman spectroscopy to study Permian gymno-spermous bisaccate pollen grains, show that these techniques can beused to

(1) analyze the same individual specimen, providing mutually rein-forcing information;

(2) image palynomorphs at appreciably greater spatial resolutionthan that afforded by optical microscopy alone;

(3) provide a basis for assessment of taxonomically distinctive charac-ters including the ornamentation of sacci and, for specimens thattransect a thin section surface, the sub-structure of mural exine;

(4) yield three-dimensional images that can be rotated inmultiple di-rections, permitting specimens to be viewed from any perspective(Supplementary video); and

(5) document the carbonaceous (kerogenous) composition of the fos-sils, the geochemical maturity of their kerogenous components,and the mineralogy of their encasing matrix.

Because all of the techniques used here are non-intrusive and non-destructive, they can be applied to especially valuable specimens suchas those archived in museum collections. And because their combineduse to analyze palynomorphs in petrographic thin sections can documentthe preservational context of such fossils, they can provide informationabout paleoenvironment and taphonomy not available from acid macer-ates. Studies of permineralized palynomorphs in situ, rather than of spec-imens isolated by acid maceration, are notably advantageous.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.revpalbo.2016.03.004.

Acknowledgments

We thank CAPES (Coordenação de Aperfeiçoamento de Pessoal deNível Superior) for supporting the 2014 visit to Brazil by J.W.S. duringwhich this study was initiated, and Barbara Calavazzi of the Central Ana-lytical Facility of the Faculty of Science, Department of Geology, Universityof Johannesburg, South Africa, for providing the SEM image in Plate I, 2.C.P.C. was supported by FAPESP (Fundação de Amparo à Pesquisa doEstado de São Paulo) Project 2010/5119-0; A.K.G., by a UCLA-Eugene V.Cota-Robles Fellowship and CSEOL (the UCLA Center for the Study ofEvolution and the Origin of Life); and A.B.K., by CSEOL and WARC (theUniversity of Wisconsin Astrobiology Research Consortium).

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