Annals of Botany 78 : 83–94, 1996
Studies of Fossil and Modern Spore Wall Biomacromolecules using 13C Solid State
NMR
A L A N R. H E M S L EY*, A N D R E W C. S C O T T†, P A T R I C K J . B A R R I E‡§
and W I L L I A M G. C H A L O N ER†
* Department of Earth Sciences, UniŠersity of Wales, Cardiff, PO Box 914, Cardiff, CF1 3YE, † Department of
Geology, Royal Holloway UniŠersity of London, Egham, Surrey, ‡ Department of Chemistry, UniŠersity College
London, Christopher Ingold Laboratories, 20 Gordon Street, London, WC1H 0AJ, UK
Received : 7 September 1995 Accepted : 22 January 1996
A range of Carboniferous lycophyte megaspore exines have been investigated using "$C magic-angle spinning nuclear
magnetic resonance (MAS NMR) spectroscopy. Their composition differs considerably from sporopollenin obtained
from an extant lycophyte. The differences observed result in part from varying degrees of diagenesis.
Fossil fern spores, gymnosperm megaspore-membranes and pollen have also been examined. These show a similar
composition to the fossil lycophyte megaspores. The constituent material of all of these exines differs considerably
from the sporopollenin obtained from comparable extant samples. Despite the changes in composition observed on
fossilisation, differences in composition between the major groups of plants may be preserved to some extent in the
fossil material. Walls of the fossil prasinophycean algal cyst Tasmanites have been examined and these show a greater
similarity to fossil cuticle and algaenans than to sporopollenins.
The effect of oxidative maceration on fossil and modern sporopollenins has also been investigated. The main
influence of oxidative maceration is the removal of unsaturated carbon environments such as aromatics ; this causes
fossil spores to be more susceptible to oxidative maceration than the modern exines. Heating of modern exine material
models the alteration of exines by diagenesis. The changes that occur on heating an extant sample to 150–225 °C give
a chemical composition that is similar to those of the fossil sporopollenins. # 1996 Annals of Botany Company
Key words : "$C solid state NMR, spores, pollen, fossil, Carboniferous lycopsids, ferns, pteridosperm, gymnosperm,
oxidative maceration, heating, thermal maturation.
INTRODUCTION
Sporopollenin is the main biomacromolecule present in the
outer wall (exine) of spores and pollen and its composition
has long been a topic of interest. The present research was
designed to investigate the chemical composition of a range
of fossil and modern spore wall materials using "$C solid
state NMR spectroscopy as the principal characterization
technique. This work follows from an initial study (Hemsley
et al., 1992) of fossil and modern sporopollenins and
pursues some of the questions raised in the earlier article.
Each "$C nucleus in a sample resonates at a slightly
different frequency when placed in a strong magnetic field
depending on its chemical environment, and the resulting
chemical shifts for different functional groups are now wellestablished. High-resolution NMR spectra may be obtained
in the solid state by rapid rotation of the sample at an angle
of 54±7° to the direction of the applied magnetic field (socalled magic angle spinning, MAS), and this technique has
been widely used to study the composition of fossil fuels and
precursor materials (Axelson, 1985 ; Wilson, 1987).
There are a number of questions concerning the chemical
composition of fossil and modern sporopollenins that it was
§ Present address : Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK.
0305-7364}96}070083­12 $18.00}0
hoped would be clarified by the application of "$C solid state
NMR spectroscopy. One major topic of interest is the extent
to which the NMR spectrum might provide a ‘ fingerprint ’
characteristic of a plant’s systematic status, as this might
prove particularly useful in studying fossil material of
questionable origin. For instance, among the samples
investigated are fossil algal cysts assignable to Tasmanites
(Prasinophyceae) ; these have previously been considered as
having been formed from sporopollenin (Brooks, 1971),
while comparison of the NMR spectrum with those of the
other spore wall biomacromolecules studied suggests a
closer affinity either to algaenans or fossil cuticles. In this
work we also compare the spectra of sporopollenin obtained
from microspores and megaspores (including seed megaspore-membranes) from the same species of plant in order
to see whether they have similar composition.
"$C solid state NMR spectroscopy also allows structural
changes that occur upon chemical treatment to be probed.
Oxidative maceration is frequently used in the separation of
spores from coals, and so knowledge of its effect on spore
structure is desirable, particularly as it is known that fossil
material will eventually ‘ dissolve ’ in Schulze’s solution (a
saturated solution of potassium chlorate in nitric acid, see
Traverse, 1988), or in fuming nitric acid. This work also
studies chemical changes that occur during heating in the
presence of oxygen, which may well be related to the
# 1996 Annals of Botany Company
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
84
1
2
4
3
6
5
F. 1. Lagenicula crassiaculeata, a Lower Carboniferous lycophyte megaspore. ¬66.
F. 2. L. subpilosa f. major, a similar lycopsid megaspore also from the Lower Carboniferous. ¬75.
F. 3. Zonalesporites sp. This genus of lycophyte megaspores has a range throughout the Carboniferous. ¬85.
F. 4. A Lower Carboniferous pteridosperm seed megaspore membrane. ¬40.
F. 5. Tuberculatisporites sp. Another lycophyte megaspore from the Upper Carboniferous. ¬50.
F. 6. Lycospora chaloneri. Microspores from the same species of heterosporous lycophyte cone as the megaspore illustrated in Fig. 2. ¬3050.
changes that occur during fossilisation. Of particular interest
are whether the changes in composition as a result of
diagenesis (changes that occur in sediments and trapped
organic materials prior to their consolidation) are gradual
or abrupt, at what temperature particular alterations in
composition occur, and how far diagenesis may blur the
characteristics of sporopollenin from any particular source
plant.
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
MATERIALS
Modern spores and pollen and seed membranes
Lycopodium claŠatum L. (Lycophyta) spores obtained from
a commercial source (BDH). Anemia sp. (Filicophyta)
spores obtained from plants at the Royal Botanic Gardens,
Kew, UK. Pinus sylŠestris L. (Coniferophyta) pollen
collected from local trees in Surrey. Dioon edule Lindley
(Cycadophyta) pollen obtained from plants at the Royal
Botanic Gardens, Kew, UK. Cycas circinalis L. (Cycadophyta) megaspore-membrane from seeds collected by
WGC at the Marie Selby Botanic Garden, Florida, USA.
Fossil spores, pollen and seed membranes
Zonalesporites sp. (Lycophyta, Fig. 3) from the Kaluga
lignite deposit, Kurovskaya Mine, Moscow Coal Basin,
Russia, (Vise! an, Lower Carboniferous) ; see Dijkstra and
Pie! rart (1957). Lagenicula crassiaculeata Zerndt (Lycophyta,
Fig. 1) from limestone, Foulden, Berwickshire, UK (late
Tournaisian, Lower Carboniferous) see Scott and MeyerBerthaud (1985). Lagenicula subpilosa f. major Dijkstra ex.
Chaloner [Lycophyta, (Fig. 2) from limestone, Pettycur,
Fife, Scotland, UK (Vise! an, Lower Carboniferous) see Rex
and Scott (1987)]. Lycospora chaloneri Scott and Hemsley
(Lycophyta, Fig. 6, see Scott and Hemsley, 1993 a) from a
cone of Flemingites scottii (Jongmans) Thomas and BrackHanes. Location and matrix as above. Tuberculatisporites
sp. (Lycophyta, Fig. 5) from Bed 20f, Middle Coal Measures
(shale), Swillington Brickpit, Yorkshire, UK. (Westphalian
B, Upper Carboniferous) see Scott (1978). Spores from
Botryopteris globosa Darrah (Filicophyta) from Coal Ball,
West Mineral Kansas, USA (Univ. Kansas 11072) see
Phillips (1980). Schopfipollenites sp. (Pteridospermophyta)
from Dolerotheca, Upper Carboniferous (Pennsylvanian),
Mazon Creek, Illinois, USA (PP 27832 Field Museum of
Natural History, Chicago, Illinois ; see Peppers and Pfefferkorn, 1970). Megaspore-membranes of seeds (Pteridospermophyta, Fig. 4) extracted from calcareous ash, Oxroad
Bay, Scotland, UK (Late Tournaisian, Lower Carboniferous) ; see Bateman and Scott (1990).
Fossil cuticle and algal cyst
Tasmanites sp. (algal cyst) obtained from Tasmanite Coal,
La Trobe, Mersey River, Northern Tasmania, Australia
(Permian) see Newton (1875). (RHBNC Geology Collection
R.1657). Eskdalia sp. (Lycophyta, cuticle) from the Russian
Paper Coal, Tovakova. British Geological Survey Collection
(Kidston Collection 1299) (Lower Carboniferous) ; see
Wilson (1931), Thomas (1968) and Collinson et al. (1994).
METHODS
All spores and pollen obtained from living plant material
used in this study were acetolysed using the method outlined
by Erdtman (1960). The material was saturated with glacial
acetic acid before acetolysis (2 min duration) and returned
to this after treatment. Each sample was then rinsed six
times with acetone to remove most of the acetic acid. This
85
treatment causes some acetylation of the sporopollenin to
occur, resulting in an increase in peak intensities at 21 and
173 ppm of the "$C NMR spectrum (see Hemsley, Barrie
and Scott, 1995 ; Figs 7a and 9a therein).
Fossil material from calcareous matrices was extracted
using 10 % HCl, picked by hand and stored in acetone
before drying. Material from siliceous samples was extracted
by maceration with 10 % HF, washed thoroughly with
water, picked and stored in acetone. Material from the
Moscow lignite was macerated gently with concentrated
HNO to facilitate release from the matrix and thereafter
$
treated as above.
Some samples of Lycopodium and Lagenicula were
subjected to oxidation by treatment with concentrated
HNO saturated with KClO (Schulze’s solution) for 30 min.
$
$
The Lycopodium was initially introduced to a small quantity
of dilute HNO (2 ml) before making up to 50 ml with the
$
above solution. This procedure was necessary to reduce the
problem of the highly exothermic reaction on initial contact
with the full strength solution. After oxidation, samples
were rinsed with water, then with acetone a number of
times, and finally air dried in a desiccator.
The samples to be subjected to heating were prepared for
study, dried and placed in open crucibles in a muffle furnace
for 1 h. The chosen temperatures of 150, 225 and 300 °C
were kept constant for this period. Air was admitted to the
samples during heating.
"$C solid state NMR spectra were obtained on a Bruker
MSL-300 spectrometer using cross-polarization (CP), high
power decoupling and magic-angle spinning. Spectra were
normally accumulated overnight (16 h), and processed using
a line-broadening factor of 50–100 Hz for the modern
material, and up to 200 Hz for the fossil material. All
spectra were acquired under identical conditions, namely
using 1 ms contact time and 1 s recycle delay at spinning
speeds of 4±5–5 kHz. The "H 90° pulse was 4 µs. Chemical
shifts are given in parts per million (ppm) relative to
external TMS. In order to alleviate the problem of
overlapping spinning sidebands, the TOSS sideband suppression sequence (Dixon et al., 1982) was used. It is
recognized that both CP and TOSS are not necessarily fully
quantitative, but these experimental conditions are required
to give reasonable quality spectra in an acceptable time on
the very small amounts of fossil material available (down to
2 mg in some cases, see Hemsley et al., 1995).
Aromaticities of the fossil samples were measured by
simple integration taking the aromatic "$C environments to
resonate between 165 and 90 ppm. Before integration, care
was taken with the phase correction (so that the baseline at
" 200 ppm and ! 0 ppm was flat). For a few samples an
empirical polynomial baseline correction was then applied.
RESULTS
Lycophyte spores (Fig. 7)
Figure 7 illustrates six NMR spectra obtained from samples
of sporopollenin of lycophyte origin. Extant Lycopodium
(Fig. 7 a) exhibits a spectrum which we have found to be
typical of a sporopollenin (Hemsley et al., 1993) and which is
86
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
a: Lycopodium
clavatum spores
b: Zonalesportes sp.
c: Lagenicula
crassiaculeata
d: Lagenicula
subpilosa f. major
e: Lycospora chaloneri
f: Tuberculatisporites sp.
200
0
100
– C–O
–
–
C=C
–
C=O
–
PPM
– C–H
F. 7. "$C NMR spectra obtained from living (a) and fossil (b–f) lycophyte spores. The spectrum from living Lycopodium exhibits a diversity of
chemical environments, whilst those obtained from fossils show only aromatics and aliphatics with some minor variation in these.
similar to that illustrated by Guilford et al. (1988). Each
distinct "$C chemical environment gives rise to a peak at a
characteristic chemical shift. The peaks can loosely be
assigned according to spectral region : 10–50 ppm indicates
aliphatic hydrocarbon environments ; 50–105 ppm indicates
oxygenated aliphatic groups ; 110–160 ppm indicates unsaturated carbon environments (olefins}aromatics) ; 160–
180 ppm indicates carboxylate groups (acids}esters).
The remaining spectra shown in Fig. 7 b–f, have all been
obtained from fossils. These differ considerably in composition from the untreated Recent spore wall material
(unlike the IR sporopollenin spectra of Brooks, 1971) but
are relatively similar to each other. The lower four spectra
(Fig. 7 c–f) are remarkably consistent despite differing in
sedimentary history and diagenetic influence. Lagenicula
crassiaculeata and L. subpilosa f. major (Fig. 7 c and d) are
both from calcareous rocks and the spores possess excellent
three-dimensional preservation (see Figs 1 and 2). Despite
this, as with the other fossil sporopollenins discussed below,
the exine material has undergone substantial modification
involving relocation or loss of oxygen and an increase in
aromatic content (in a process akin to coalification). Hence
the spectra consist of two broad peaks corresponding to the
aromatic and aliphatic carbons. The aromatic peaks are
clearly compound with contributing peaks at 126, 138 (due
to bridgehead carbons) and 152 ppm (due to phenolic
carbons).
Lycospora chaloneri (Figs 6 and 7 e) are the microspores
produced within the same cone as Lagenicula subpilosa f.
major and these give a similar "$C spectrum as does the
sample of Tuberculatisporites (megaspore of Sigillaria, Figs
5 and 7 f). The Tuberculatisporites result is important as the
sample was extracted from a siliceous shale, unlike the other
fossil samples discussed so far. Aromaticity values (the ratio
of aromatic carbon to total carbon content) measured from
the NMR spectra are give in Table 1. It thus appears that
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
87
T     1. Measured aromaticities of the fossil samples. (Aromaticity ¯ aromatic peak area}total carbon area)
Sample
Zonalesporites
Lagenicula crassiaculeata
L. subpilosa f. major
Tuberculatisporites sp.
Lycospora chaloneri
Botryopteris globosa
Schopfipollenites sp.
Seed megaspore membrane
Tasmanites
Eskdalia
Aromaticity %
Organ
37
43
48
50
53
56
45
33
15
10
Carb. lycopsid megaspore
Carb. lycopsid megaspore
Carb. lycopsid megaspore
Carb. lycopsid megaspore
Carb. lycopsid microspore
Carb. fern meiospore
Carb. pteridosperm pollen
Carb. pteridosperm seed
Permian algal cyst
Carb. lycopsid cuticle
a: Anemia sp. pollen
b: Botryopteris sp. pollen
200
0
– C–O
–
–
C=C
–
C=O
–
100
PPM
– C–H
F. 8. "$C NMR spectra obtained from a living fern spore (a) and a fossil fern spore (b). The living spore spectrum shows a distinctly different
composition from that of Lycopodium whilst the fossil fern has a spectrum similar to the fossil lycophytes, but with a relatively higher aromatic
signal.
the diagenetic change produced in the sporopollenin is
independent of the nature of the matrix in the samples
examined here.
Zonalesporites (Figs 3 and 7 b) differs from the four fossil
spores mentioned above in retaining evidence of the
oxygenated aliphatic (55–95 ppm) and carboxyl peaks
88
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
a: Dioon edule pollen
b: Schopfipollenites sp. pollen
c: Cycas circinalis
megaspore-membrane
d: Fossil pteridosperm
megaspore-membrane
100
200
0
– C–O
–
–
C=C
–
C=O
–
PPM
– C–H
F. 9. "$C NMR spectra of living gymnosperm pollen and a megaspore membrane (a and c) and their fossil equivalents (b and d). In both cases,
the fossil material is greatly altered by diagenesis, but in the case of the seed membranes, the lower aromatic component (compared with the pollen)
is retained by the fossil.
(160–180 ppm) present in the extant lycophyte sporopollenin
(Fig. 7 a). This fossil material has been subjected to partial
oxidative maceration which might affect composition
slightly. However, from the results given below, the effect of
this treatment is unlikely to alter these two additional
features. This example suggests that there is less corruption
of the chemical identity of the original sporopollenin
retained in material of very low rank (i.e. not subjected to
high temperatures associated with deep burial) .
Fern spores (Fig. 8)
The spectrum obtained from the extant fern spores is
different from that of Lycopodium. Most noticeable is the
significant reduction in the number of aliphatic carbon
environments (compare the region 20–35 ppm in Figs 7 a
and 8 a). The lower proportion of aliphatic material present
in the extant fern spores may be retained to some degree
during diagenesis, as manifest by the spectrum of fossil fern
spores (Fig. 8 b), in which the aromatic peak intensity is
higher than that of the aliphatic peak (unlike those of the
fossil lycophytes shown in Fig. 7). This results in the higher
aromaticity values for the fossil fern spores presented in
Table 1, and this may provide a basis by which spores of
ferns might be distinguished from those of lycophytes. It
should, however, be borne in mind that aromaticity may
also be affected by thermal history as is demonstrated
below.
Gymnosperm pollen and seed megaspore-membranes (Fig.
9)
Gymnospermous plants produce sporopollenin in the
form of a pollen wall, and also as the seed megaspore
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
89
a: Tasmanites sp. cyst
b: Fossil cuticle
200
0
– C–O
–
–
C=C
–
C=O
–
100
PPM
– C–H
F. 10. "$C NMR spectra of fossil prasinophycean algal cysts (a) and cuticle of Eskdalia, a fossil lycophyte (b). Both spectra illustrate the higher
aliphatic proportions within these two biomacromolecules (algaenans, cutans ; see Collinson et al., 1994) which are very different from fossil
sporopollenins.
membrane around the female gametophyte. This latter
structure is interpreted as the relictual megaspore wall,
retained from an evolutionary precursor in which the
megaspores were freely dispersed (Chaloner and Pettitt,
1987).
The NMR spectrum obtained from pollen (Fig. 9 a) from
Dioon edule (a cycad) illustrates a spectrum that appears
intermediate between the lycophyte and fern spores. In this
material, the unsaturated carbon environments are rather
less clearly defined and form less of the sporopollenin
composition than in the ferns or lycophytes. A fossil
gymnosperm pollen (a medullosan Schopfipollenites) exhibits a spectrum similar to that of the fossil lycophytes with
a comparable aromaticity (Fig. 9 b).
Extant cycad seed megaspore-membranes show a spectrum (Fig. 9 c) similar to that produced by the extant pollen,
but a number of additional oxidized aliphatic components
are present (65 and 99 ppm) whilst the peak at 52 ppm
(present in the pollen) is absent. The fragility of these
extremely thin sporopollenin membranes and their degree
of attachment to the surrounding seed wall layers, makes it
difficult to be certain that all of the non-sporopollenin
material has been removed even after acetolysis. It is
possible that some of the differences between the megaspore
membrane and the pollen are a result of retention of some
of these adjacent layers in the former.
Despite this potential problem, it is clear that the higher
proportion of aliphatics present in the extant membrane
occurs also in the fossil material of a pteridosperm seed
megaspore-membrane (Fig. 9 d) which has an aromaticity of
only 33 %. This observation lends support to the suggestion
that the relative proportion of aromatics to aliphatics is
90
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
a: Lycopodium clavatum spores
b: Lycopodium
clavatum spores
after oxidative
maceration
c: Lagenicula crassiaculeata
d: Lagenicula crassiaculeata
after oxidative maceration
100
200
0
– C–O
–
–
C=C
–
C=O
–
PPM
– C–H
F. 11. "$C NMR spectra illustrating the effects of oxidative maceration on both living (a and b) and fossil (c and d) spores. Unoxidised material
(a and c) loses components within the aromatic range (105–150 ppm) upon oxidative maceration (b and d). Fossil material (d) loses proportionally
more of these components and this accounts for the observed destructiveness of this technique.
retained to some degree in the fossil material despite
diagenesis.
The spore wall of Tasmanites (Fig. 10)
Figure 10 a illustrates the "$C NMR spectrum obtained
from the Tasmanites sp. cyst wall. Members of this genus
have been attributed to the algal group Prasinophyceae
(Wall, 1962), now Pterospermales (Chlorophyta) (GuyOhlson and Boalch, 1992), and were reported to consist of
sporopollenin by Brooks and Shaw (1971) who found that
the infra-red spectrum from their wall material was the same
as that from spores and pollen. From our spectra it is
immediately evident that of Tasmanites is very different
from the NMR spectra of both the modern and fossil spores
and pollens. The bulk of the material is aliphatic with only
a small proportion being of aromatic composition. This
corresponds quite closely to a spectrum obtained from an
alga (Zelibor et al., 1988) or a fossil cuticle (Fig. 10 b), in this
case that of Eskdalia, a lycophyte cuticle from the Lower
Carboniferous of Russia. (Collinson et al., 1994).
This result strongly suggests a closer similarity of
tasmanite walls to algal or cuticular material as opposed to
sporopollenin. This striking difference in composition
suggests that NMR characteristics should distinguish
between prasinophycean algal cysts and the sporopollenin
of archegoniate plants.
OxidatiŠe maceration of spores (Fig. 11)
Figure 11 illustrates four spectra, two obtained from
acetolysed Lycopodium and two from the fossil Lagenicula
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
91
a: Pinus sylvestris pollen
b: Pinus sylvestris pollen
heated to 150°C
c: Pinus syvestris pollen
heated to 225°C
d: Pinus sylvestris pollen
heated to 300°C
200
0
100
– C–O
–
–
C=C
–
C=O
–
PPM
– C–H
F. 12. "$C NMR spectra showing the effects of heating (for 1 h) on the chemistry of extant pollen walls. Acetolysed pine pollen (a) shows a typical
spectrum, but as this is heated (b to d) the spectrum begins to resemble (at low temperatures) low rank fossil material (b) and at higher
temperatures, greatly altered material (c and d). (Compare b to Fig. 7 b, and c to Fig. 7 c–f).
crassiaculeata. The unoxidized samples (Fig. 11 a and c) are
the same as those in Fig. 7 and are repeated here for
immediate comparison with the oxidatively macerated
material. Macerated Lycopodium (Fig. 11 b) clearly illustrates that the main effect of this treatment is the removal
of the unsaturated carbon environments which resonate
between 110 and 150 ppm. The spectrum is barely altered in
other respects.
The spectrum of oxidised fossil sporopollenin (Fig. 11 d)
shows a significant loss of aromatic material (120–160 ppm)
as a result of the treatment. The relatively poor signal-tonoise ratio is a result of the very small amount of sample
analysed that survived the macerative treatment. It can be
seen that it is the unsaturated carbon environments that are
attacked by oxidative maceration in both modern (Fig. 11 b)
and fossil (Fig. 11 d) sporopollenins, but that proportionally
more material is lost from the fossil (in this case, aromaticity
decreases from 48 to 26 %). Benzoic acid was recovered
from the oxidizing solution which is a likely derivation from
the aromatic component of the sporopollenin.
Modelling diagenesis by heating
Acetolysed pine pollen (Fig. 12 a) was heated for 1 h at
150 °C (Fig. 12 b), 225 °C (Fig. 12 c) and 300 °C (Fig. 12 d)
in each case. The resulting spectra show considerable
changes from those of the unheated pollen sample. The
sample subjected to 150 °C shows four main peaks (29, 71,
129, 170 ppm). Homogenization of both the aliphatic and
aromatic components has occurred resulting in the loss of
fine structure from the spectrum. A small oxygenated
aliphatic peak is retained but the bulk of oxygenated
92
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
carbons are lost. The carboxyl peak is diminished but
clearly recognizable. This spectrum resembles that obtained
from the low rank Zonalesporites (Fig. 7 b).
After heating at 225 °C (Fig. 12 c), only a single aliphatic
and aromatic peak remain and the spectrum closely
resembles those from most of the fossil samples investigated ;
the observed aromaticity is 54 %.
The sample heated at 300 °C (Fig. 12 d) shows a major
change from the above spectra. Here, virtually all of the
aliphatic component has been lost and the remaining
material is almost entirely aromatic in composition as seen
in fossil samples significantly affected by diagenesis (i.e.
above the lignite rank, Davis et al., 1988).
DISCUSSION AND CONCLUSIONS
The above results provide information regarding the
effects of diagenesis on the chemistry of spore and pollen
walls. It is evident that exine material from vascular plants
differs only slightly between certain major groups (compare
the Lycopodium, Anemia, Dioon, Cycas and Pinus) and that
the differences observed previously may have been influenced by different preparatory treatment of the samples
(Guilford et al., 1988 ; Hemsley et al., 1992, 1993).
The fossil material is considerably altered from the
original sporopollenin, exhibiting a redistribution or loss of
oxygen and a significant increase in aromatic content. The
results from the diagenetically altered spores are very
similar to sporinite samples investigated by Axelson (1985)
and Davis et al. (1988). In none of the fossil sporopollenins
are there clear indications of any of the distinctive
characteristics of the original sporopollenin that would
enable us to make a close taxonomic assignment. Despite
this, differences in aromaticity of fossil sporopollenins may
provide the basis for assignment to a major plant group.
Low rank material may retain more information but even
this is lacking in distinctive characteristics. There are no
apparent differences between the exine material derived
from micro- and megaspores from the same plant, suggesting
that no change in the plants synthesis of sporopollenin has
accompanied the evolution of distinct, separate male and
female morphology.
A feature of the spectra of fossil vascular plants is that the
observed aromaticity is about 50 % which is similar to that
of a low-rank coal. Only in the fossil megaspore-membrane
does the aromaticity differ greatly from 50 % and is perhaps
more similar to the cuticular material of Eskdalia.
NMR analysis of tasmanite cysts clearly demonstrates a
closer affinity with algaenans and cuticular material (Eskdalia). The observed aromaticity is far lower than that of the
sporopollenins. This ratio is evidently characteristic for
these two different groups of biomacromolecule.
Oxidative maceration of sporopollenins results in the
removal of unsaturated carbon environments in the range of
110–150 ppm, both in modern and fossil material. However,
in the fossil material (prior to oxidative maceration) the
aromatics within the appropriate range account for a much
greater proportion of the exine material. This greater
proportion of susceptible material explains why fossil spores
can be ‘ dissolved ’ in Schulze’s solution whilst modern
Lycopodium retains its integrity for a considerably longer
period under the same conditions.
Pine sporopollenin heated in the presence of air shows a
number of striking differences from the acetolysed, unheated
sample. The sample heated to 150 °C (Fig. 12 b) shows a
remarkably similar spectrum to that obtained from the low
rank Zonalesporites (Fig. 7 b) suggesting that this degree of
heating is a reasonable model for the diagenesis experienced
by such a sample. One of the first effects of heating seems to
be the homogenization of the aromatic peaks into a single
broad-based peak. This has also been observed at the lower
temperature of 100 °C during an acetolysis treatment lasting
for 3 h as opposed to the 2 min of acetolysis used here
(Hemsley et al., 1993). Hayatsu et al. (1988) illustrate NMR
spectra of heated sporopollenins and their sample subjected
to 150 °C for 3 months is similar to that shown here. It is
apparent that, beyond a critical interval, the length of time
for which the sample is heated is of no importance. More
critical is the maximum temperature attained. Heating to
225 °C clearly approximates the degree of diagenesis
experienced by the majority of fossil material shown here
whilst heating to 300 °C has a dramatic effect on the
sporopollenin, removing or altering almost all of the
aliphatic component and probably simulating an extreme
case of diagenesis. If changes in fossil sporopollenin
attributed to diagenesis are indeed the result of past heating,
it may be possible to ascertain, on the basis of an NMR
spectra, the maximum temperature attained.
An important observation is that the effect of heating
sporopollenin in air is completely different from that of
oxidative maceration ; the former increases the aromatic
proportion of the substance in question while the latter
causes this to diminish.
Diagenetic alteration of sporopollenin and spore colour
There has been significant interest in the alteration of
sporopollenin (spores, sporinite) during diagenesis especially
with regard to thermal alteration. Several authors have
noted both physical and chemical changes in sporopollenin
during natural and artificial thermal maturation (e.g.
Durand, 1980 ; Sengupta, 1980 ; Pie! rart, Postiau and Roism,
1980 ; Schenck et al., 1981). The NMR results presented
here indicate particularly that aliphatic groups are lost or
reorganized and that aromatic groups increase (relatively)
during thermal alteration. Whilst there has often been
significant chemical alteration of the sporopollenin there
appears, at least at moderate temperatures, to be less
alteration in the spore wall ultrastructure (Scott and
Hemsley, 1993 b) despite changes in the overall dimensions
of the palynomorph (e.g. Pie! rart et al., 1980). For the most
part, ultrastructure appears to be affected more by pressure
and lithology than moderate heating (Scott and Hemsley,
1993 b).
One of the most obvious physical changes in spores
caused by thermal maturation is that of a change in colour
(reviewed by Marshall, 1991). It is generally recognized that
spores change from colourless to yellow, orange, brown and
finally black with increasing thermal maturation. This
phenomenon has been widely used for interpreting the
Hemsley et al.—Spore Wall Analysis by "$C Solid State NMR
degree of maturation of kerogen in a rock (Durand, 1980).
Many groups of palynomorphs have been used in this way
and some colour scales use not only spores (sensu lato), but
cysts of dinoflagellates and acritarchs (the latter used in preDevonian assemblages).
The results of our NMR study suggest that there may be
problems in assuming that colour changes in other groups
of palynomorph bear any constant relationship to colour
changes in archegoniate spores. The initial chemical composition of the resistant walls are different from those of at
least one kind of planktonic cyst, and changes during
diagenesis are likely to be equally different. There is no
reason to suppose that the degree of colour change in an
organic wall made of sporopollenin (containing both
aliphatic and aromatic components) and of algaenan (largely
aliphatic components) is the same. However, if the same
kind of palynomorph is compared, i.e. spore, pollen or
acritarch, then results are probably comparable (van Bergen
and Kerp, 1990). We suggest therefore that care be exercised
in equating spore and algal (tasmanite, dinoflagellate and
acritarch) colour changes. This may be of particular
importance in pre-Silurian rocks where there is no vitrinite,
which means that comparisons with a standard reflectance
scale cannot be made.
The darkening in colour of spores and pollen during
diagenesis has resulted in the necessity of ‘ cleaning ’ them
(i.e. making them less opaque) for light microscopic studies.
Dark brown or black samples are generally oxidised to a
lighter colour. A significant problem has concerned very
dark spores which, when oxidatively macerated, disintegrate
(Marshall, 1980). In the cases of spores which have
undergone intense diagenesis (maturation) aromatic structures dominate. When attacked with an oxidising acid, the
subsequent removal of aromatics leaves insufficient material
to retain the structure, causing it to fall apart. We believe
therefore, that no chemical technique is likely to cause very
mature spores to revert to yellow whilst still remaining
intact (without special measures ; Marshall, 1980).
A C K N O W L E D G E M E N TS
We thank the British Geological Survey (Eskdalia), M.
Oshurkova (Zonalesporites), C. Maples and the staff of the
herbarium, University of Kansas (Botryopteris), P. Crane,
Field Museum of Natural History, Chicago (Schopfipollenites), the Royal Botanic Gardens, Kew, UK, and the Marie
Selby Botanic Garden, Florida, for the supply or loan of
material. We also thank D. Butler for running many of the
samples on the ULIRS solid state NMR facility at UCL.
This work was supported by a grant under the NERC
biomolecular palaeontology special topic (to WGC and
ACS), for which we record our grateful thanks.
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