Tissue & Cell 36 (2004) 263–273
Ultrastructure of Early Jurassic fossil plant cuticles:
Pachypteris gradinarui Popa
G. Guignard a,∗ , M.E. Popa b,1 , G. Barale a,2
a
Paléobotanique, Université Claude-Bernard Lyon 1 and UMR 5125 CNRS, Bâtiment Darwin A, 7 Rue Dubois, 69622 Villeurbanne Cedex, France
b Laboratory of Palaeontology, Faculty of Geology and Geophysics, University of Bucharest, 1, N. Balcescu Ave., 70111 Bucharest, Romania
Received 19 December 2003; received in revised form 13 March 2004; accepted 1 April 2004
Abstract
Exceptional preservation of extinct Pachypteris extra-epidermal cuticle enabled the first detailed statistical measurements of its ultrastructure using transmission electron microscopy. Pachypteris is a leaf genus of the Mesozoic belonging to seed fern foliage of the order
Corystospermales. The species studied in this paper is Pachypteris gradinarui Popa [Rev. Palaeobot. Palynol. 111 (2000) 31], based on
fossils which are Early Jurassic in age (Hettangian-Sinemurian, approximately 205–190 million years old). Both the upper and the lower
cuticles were thoroughly examined, including the detail of the stomatal complexes and epidermal cells. The data obtained from our TEM
analysis, together with the confidence intervals, were very useful to give precise description of the cuticles as they distinguished between
upper and lower epidermal and stomatal cell types. Moreover a combination of characters was used to develop the first dichotomous
key based on ultrastructural characters, i.e. not only the total thickness of the cuticle but also details and proportions of A cuticle proper
and B cuticular layer. Comparisons with ultrastructures known from other Pachypteris species show that the influence of space and time,
diagenetic processes, and/or processes related to technical procedures, seem to be minimal within this genus. Detailed studies of this type
may be very useful for further comparisons among other species and at higher taxonomical ranks.
© 2004 Elsevier Ltd. All rights reserved.
Keywords: Ultrastructure; Fossil leaf cuticles; Pachypteris gradinarui; Pteridospermopsida; Early Jurassic; South Carpathians
1. Introduction
Cuticle is a very thin film covering the epidermis of the
aerial parts of many tracheophytes. It is a very precise and
even perfect external moulding of epidermal cells, and as
it is often the only organic remains of fossil plants in various types of sediments their detailed study is very useful in Palaeobotany. Cuticles have been studied for decades
(Harris, 1956; Archangelsky, 1991) and high variation has
long been observed with light (LM) and scanning electron
(SEM) microscopes. Ultrastructural studies using a transmission electron microscope (TEM) are still not in common
practice, although they have been used for extant plant cuticles for approximately 40 years (Holloway, 1982). Thanks
∗ Corresponding author. Tel.: +33-4-7244-8203;
fax: +33-04-7244-8203.
E-mail addresses: guignard@univ-lyon1.fr (G. Guignard),
mihai@mepopa.com (M.E. Popa), barale@univ-lyon1.fr (G. Barale).
1 Tel.: +40-72-273-4070; fax: +40-21-211-3120.
2 Tel.: +33-4-7244-8203; fax: +33-4-7244-8203.
0040-8166/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tice.2004.04.002
to very fine details observed within ultrathin sections of cuticles using TEM and found as very useful compared with
LM or SEM approaches, the ultrastructural studies on fossil
cuticles began in 1986 with the works of Archangelsky and
Taylor, and Archangelsky et al.
The Mesozoic seed fern group Corystospermales, belonging to class Pteridospermopsida (Taylor and Taylor, 1993;
Stewart and Rothwell, 1993), represents a very interesting
plant group when analysing the mixture of both primitive
(e.g. compound leaves similar to fern fronds) and evolved
characters (e.g. complex reproductive structures such as
Umkomasia), along with the palaeogeographic significance
(Vakhrameev, 1991) and the phytogeographic importance
of many of their representatives. Among seed fern ultrastructural studies that have been reported (Archangelsky
et al., 1986; Taylor et al., 1989; Maheshwari and Bajpai,
1996a,b; Bajpai, 1997), some studies have focused on the
Corystospermales genus Pachypteris (Labe and Barale,
1996; Baldoni and Barale, 1996; Bajpai and Maheshwari,
2000). However, their results were mostly qualitative, i.e.
description of the structures, and dealt only with epidermal
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G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
cell outlines not including stomatal apparatus, using lower
magnifications with mainly short descriptions and measurements, and represent the current state of our knowledge of cuticular ultrastructure in Pachypteris. The genus
Pachypteris Brongniart emend. Harris (Brongniart, 1828;
Harris, 1964) is a common form genus of pteridosperm (seed
fern) foliage for Triassic-Jurassic deposits of the Northern Hemisphere. Lower Jurassic (Hettangian-Sinemurian,
approximately 205–190 million years old) continental deposits of the South Carpathians, central Romania, yield a
rich and well preserved compression flora, including an
abundant pteridosperm assemblage of which Pachypteris
is represented by several species. Pachypteris gradinarui
Popa (Popa, 2000), a new established taxon was collected
from Cristian, an important fossil plant locality in Romania
due to its excellently preserved fossil plants. Using results
from these prior LM and SEM studies (Popa, 2000), and
taking advantage of the high preservation quality allowing
detailed observations of not only cuticle located above ordinary epidermal cells, but also above guard and subsidiary
cells of the stomatal apparatus, we examined Pachypteris
cuticle at the ultrastructural level using TEM for this study.
2. Material and methods
The leaf fragments (Fig. 1) occur on sandstone hand
specimens collected from Cristian (Brasov County, South
Carpathians, Romania) (Popa, 2000). The sandstone is a fine,
lithic sandstone, Lower Jurassic, Hettangian-Sinemurian in
age. The leaf compressions preserve the frond rachis and
pinnules with their diagenetically altered mesophyll, cell
wall residues, and very well preserved cuticle including the
details of epidermal cells and stomatal complexes.
The cuticles were obtained by treating leaf compressions
with Schultze’s Reagent (HNO3 + KClO3 ). KClO3 crystals
and concentrated HNO3 acid were added to glass tubes
containing the pinnule fragments until the upper and lower
cuticle separated. After 4 weeks in paraformaldehyde solution, cuticles were prepared for the TEM study. This method
is described in the papers of Lugardon (1971) and Guignard
et al. (2001), including embedding and staining the material.
Cuticles were pre-stained with osmium-tetroxide, embedded
in blocks of Epon resin, cut with a diamond knife (Reichert
Ultracut S) and finally stained with uranyl acetate and lead
citrate. Two resin blocks, one containing the lower cuticle
Figs. 1–31. Different views of the cuticle of Pachypteris gradinarui. Except Figs. 1 (camera microphotograph) and 18–20 (light microscope microphotographs), all other figures are transmission electron microscope microphotographs. OP: outer part of the cuticle; IP: inner part of the cuticle; EC: location
of an ordinary epidermal cell; AW: anticlinal wall between two cells cuticle; CW: cell wall residues; SP: stomatal pit of the stomatal apparatus; SC:
subsidiary cell location; GC: guard cell location. Some lettering indicate the different parts of the cuticles: cuticle proper A (= A1 (upper A1U + lower
A1L for upper ordinary epidermal cell cuticle only) + A2) + cuticular layer B. B layer is making a reticulum with very various schemes observed in
each type of cell; thus, in order to give the most complete view of all schemes observed, different examples are given for each type of cell. For the
same reasons, the cuticles of all types of cells consisting of layers similar in ultrastructure, in order to give a better overview of each layer detailed
figures at different magnifications (up to 125,000×) are provided. Fig. 1. General view of a leaf, No. G44. Figs. 2–10. Ordinary epidermal cell upper
cuticle. Fig. 2. General view. The outer part is delimited with a hardly visible layer (arrows) at this low magnification (compare with Fig. 11). Numbers
correspond to figures detailing parts of the cuticle in this page, parallel section, No. GGE978, 1800×. Fig. 3. Outermost part, made up with thin upper
A1U and thicker lower A1L zones, parallel section, No. GGE981, 14,500×. Fig. 4. The above part of the anticlinal wall is made up with very contrasted
B layer fibrils making a pillar-shape structure, parallel section, No. GGE973, 60,000×. Fig. 5. Outermost A1 layer, composed of sparsely lamellate
A1U zone covering dispersed material, perpendicular section, No. GM0174, 35,000×. Fig. 6. A2 granular layer, perpendicular section, No. GM0169,
125,000×. Fig. 7. B fibrilous layer arranged in a reticulum, parallel section, No GGE970, 28,000×. Fig. 8. Cell wall residues with very parallel lines
(see Figs. 17, 27, and 31 for comparison), parallel section, No. GGE965, 100,000×. Fig. 9. Fibrils of B layer are arranged as herring bones (arrows),
parallel section, No. GGE974, 17,000×. Fig. 10. Fibrils of B layer are arranged in a reticulum with very condensed and very clear spaces, perpendicular
section, No. GMD199, 45,000×. Figs. 11–17. Ordinary epidermal cell lower cuticle. Fig. 11. General view of the cuticle. In this case, the outermost
part is clearly delimited (compare with Fig. 2). Numbers correspond to figures detailing parts of the cuticle in this page, parallel section, No. GGE983,
1400×. Fig. 12. Outer part of the cuticle, with the cuticle proper (A1 + A2) and the reticulum of the cuticular layer B, parallel section, No. GGE990,
28,000×. Fig. 13. Outermost continuous A1 polylamellate layer, covering the A2 granular layer, perpendicular section, No. GM0468, 60,000×. Fig. 14.
Outermost part with an interrupted A1 polylamellate layer on the right side, parallel section, No. GGE993, 125,000×. Fig. 15. Reticulum of B layer
leaving empty spaces different in size, parallel section, No. GGE994, 28,000×. Fig. 16. The fibrils of the B layer show a very condensed structure,
perpendicular section, No. GM0331, 28,000×. Fig. 17. Parallel and numerous lines inside the cell wall running around the cell location (compare with
Figs. 8, 27, and 31), perpendicular section, No. GM0339, 45,000×. Figs. 18–20. Stomatal apparati cells cuticle, general views. The sections show
different aspects according to their location, one or two guard cells cuticles being visible depending on the orientation of the sections, the stomatal pit
being more or less opened. Numbers correspond to the figures detailing this part of the cuticle, perpendicular sections, light microscope, No. P1-P2-P3,
1000×. Figs. 21–27. Subsidiary cell cuticle. Fig. 21. Outer part of the cuticle, showing the cuticle proper (A1 + A2) and the fibrils of the cuticular layer
B, short and various in diameter. Numbers correspond to figures detailing parts of the cuticle in this page, perpendicular section, No. GM0450, 60,000×.
Figs. 22 and 23. Outermost part with a faintly stained polylamellate A1 layer, covering the A2 granular layer, perpendicular sections, No. GM0470 and
No. GM0471, 60,000×. Fig. 24. A2 granular layer, perpendicular section, No. GM0474, 35,000×. Fig. 25. Rather sparse fibrils are parallel and waving,
perpendicular section, No. GM0453, 75,000×. Fig. 26. Detail of Fig. 25 showing the reticulum of fibrils at a higher magnification, perpendicular section,
No. GM0454, 125,000×. Fig. 27. Innermost part along the cell location where the cell wall shows condensed lines (compare with Figs. 8, 17, and 31),
perpendicular section, No. GM0450, 60,000×. Figs. 28–31. Guard cell cuticle. Fig. 28. Boundary between a guard and a subsidiary cell cuticle, with
fibrils parallel just in the centre and making on both sides a reticulum orientated differently, perpendicular section, No. GM0456, 60,000×. Fig. 29. A1
polylamellate layer is not continuous in this outermost part, perpendicular section, No. GM0461, 125,000×. Fig. 30. B layer fibrils are running in all
directions, perpendicular section, No. GM0458, 60,000×. Fig. 31. Innermost part around the cell location with cell wall residues made with parallel lines
(compare with Figs. 8, 17, and 27), perpendicular section, No. GM0457, 60,000×.
G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
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G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
and the second the upper cuticle, were prepared and cut to
70 nm thick ultra-sections. All types of cell cuticles were
cut transversal and parallel to the length of the cells. The
ultra-sections were mounted on 300-Mesh uncoated grids
and studied with TEM. The total number of studied grids,
each containing one or several ultra-sections, depending on
their size, was 65 (perpendicular to the leaf length: 10 for
the upper cuticle and 35 for the lower cuticle; parallel to the
leaf length: 20 in total, 10 for each upper and lower cuticles). The resin blocks and negatives (called GGE or GM in
the figure captions for transmission electron microscopy, P
for light microscopy, G for camera) are stored in the Guignard Collection in Lyon and in the Gradinaru collection in
Bucharest. Cuticle preparations were illustrated using Corel
Draw ver. 7 for vectorial drawings and Corel Photopaint
ver. 7 for bitmap images. The TEM used is a Philips model
CM120 at Centre technologique des microstructures (CT␮),
Université Claude-Bernard, Lyon 1, in Villeurbanne, France.
apparatus cuticle located above guard and subsidiary cells),
and statistical values are provided (Tables 1 and 2). In most
cases, the cell wall is made up of very concentrated parallel
lines (Figs. 8, 17, 27, and 31), and were not included in the
measurements and in the ultrastructure description.
Upper cuticle: ordinary epidermal cell cuticle (Figs. 2–10;
Tables 1 and 2).
The cuticular membrane (CM) (13.7 ␮m in mean thickness) consists of a thin cuticle proper (CP) (= A, 30.5%
of the whole cuticle) and the cuticular layer (CL) (= B,
69.5%). The A1 layer, very rarely absent, is composed of a
thin, faintly stained, sparsely lamellate upper zone A1U and
a thicker mainly amorphous lower zone A1L , which more
or less contains the same material as the A1U zone (Figs. 3
and 5). The A1U zone covers the A2 granular layer (Fig. 6).
Below A2 is the B layer, which is made up of a reticulum
of fibrils arranged in very diversed schemes (Figs. 7 and
9–10, see also the other detailed B layer figures among all
other types of cells as only the whole set gives a complete
idea of B layer in each type of cell: Figs. 15–16, 25–26, and
30). Among these schemes, fibrils have in different areas
a “herring bone” appearance (termed used as in Holloway,
1982).
Lower cuticle: ordinary epidermal cell (Figs. 11–17) and
stomatal apparatus (Figs. 18–20) (subsidiary (Figs. 21–27)
and guard cell (Figs. 28–31)) cuticles (see also Tables 1
and 2).
3. Results
The terminology used is that of Archangelsky et al. (1986)
and commonly used for fossil plants cuticles. Sections observed allowed 30 measurements for each type of cell (called
ordinary epidermal cells and used for cuticle located above
normal epidermal cells, to be distinguished with stomatal
Table 1
Statistical values, made with 30 measurements for each type of cell cuticles
Ordinary epidermal cells
Upper cuticle
CM
CP (A)
A1
A1U
A1L
A2
CL (B)
OL (nm)
TL (nm)
Lower cuticle
Mean
Min.–max.
Percent
S.D.
Var
Mean
Min.–max.
Percent
S.D.
Var
13.68
4.17
1.64
0.10
1.54
2.53
9.51
7.71–23.63
2.25–6.19
0.63–3.31
0.05–0.25
0.50–3.16
1.50–4.40
3.90–19.50
100
30.48
11.99
0.73
11.26
18.49
69.52
5.03
1
0.67
0.045
0.65
0.58
4.57
25.28
0.99
0.45
0.002
0.42
0.34
20.92
12.26
0.20
0.05
6.97–23.27
0.09–0.47
0.02–0.08
100
1.63
0.41
4.89
0.09
0.02
23.91
0.08
0.003
0.15
12.06
5.80
7.59
0.06–0.40
6.80–23.00
3.90–8.40
3.90–16.70
1.22
98.37
0.08
4.83
1.92
3.52
0.07
23.29
0.004
0.01
Var
Stomatal apparatus
Subsidiary cell cuticle
CM
CP (A)
A1
A2
CL (B)
OL (nm)
TL (nm)
Guard cell cuticle
Mean
Min.–max.
Percent
S.D.
5.30
0.23
0.05
0.18
5.07
8.75
7.09
3.60–9.90
0.15–0.31
0.02–0.08
0.09–0.28
3.35–9.68
7.40–15.40
3.90–8.30
100
4.34
0.94
3.40
95.66
1.45
0.032
0.015
0.04
1.45
2.67
1.49
Var
2.10
0.001
0.0002
0.002
2.10
0.001
0.002
Mean
Min.–max.
Percent
S.D.
3.99
0.28
0.08
0.20
3.71
11.80
7.45
0.99–8.25
0.19–1.07
0.04–0.14
0.12–0.98
0.72–8.02
3.90–23.0
3.90–7.70
100
7.02
2.01
5.01
92.98
2.31
0.15
0.03
0.15
2.35
6.66
0.96
5.35
0.023
0.001
0.023
5.50
0.04
0.001
Min.–max.: minimum and maximum values observed; percent: percentage of each detailed part of the cuticle; var: variance. The cuticular membrane
CM is made up with cuticle proper CP (= A1 layer + A2 layer) and cuticular layer CL (= B layer). In the upper ordinary epidermal cell cuticle, the
A1 layer is composed of an upper A1U and a lower A1L parts. All other A1 layer cuticles are composed of lamellae, opaque OL, and translucent TL.
Except for very thin OL and TL measured in nm, all other measurements are in ␮m.
G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
267
Table 2
√
Confidence interval (= x̄ ± (var/n) × 1.96, giving 95% ␣ risk) of the different types of cell cuticles, calculated among the different layers, zones and
types of lamellae
Continuous line shows affinities between different cells types, // shows weak affinities, disruption along a line shows disaffinities.
The cuticles located just above the three types of cells
are similar in ultrastructure (12.3, 5.3, and 4 ␮m in mean
thickness, respectively), and are composed of the CP (= A
= polylamellate A1 + granular A2; 1.6, 7, and 4.3% of
the whole cuticle, respectively) and the CL (= B; 98.4, 93,
and 95.7% of the whole cuticle, respectively). The A1 layer
(Figs. 13–14, 22–23 and 29), absent in some places (Figs. 14,
23 and 29), consists of about 5–8 opaque (5.8, 15.7, and
11.8 nm in mean thickness, respectively) and translucent
(7.6, 7.1, and 7.5 nm of thickness in mean, respectively) thin
lamellae. The A2 layer is granular and homogeneous (Figs.
12–13, 22–24, and 29). The B layer is composed of a reticulum of fibrils also arranged in very diversed schemes (Figs.
15–16, 25–26, and 30).
4. Discussion
4.1. General considerations, quantitative new data and
their importance
Following LM and SEM study of this taxon (Popa, 2000),
TEM results support the exceptional quality of the fossil
material, as many ultrastructural details could be observed.
Despite the age of the compressions, the cell wall residues
are usually in the lowermost part of the cuticle, and the
quasi-omnipresent outermost A1 polylamellate layer of the
cuticle is observed. Thickness measurements and other comparisons among the types of cuticles covering different types
of cells were possible. Although fragility of the cuticles
among several pteridosperm taxa has been already noted and
discussed (Kerp and Barthel, 1993; Krings and Kerp, 1997),
stomatal apparati were easily observed and measured in P.
gradinarui. Stomatal complex detail is usually difficult to
obtain with TEM and is often rarely observed in fossil plant
cuticles. They are not provided in the papers of the other
authors discussed below, certainly due to both rarity in the
cuticles compared to ordinary epidermal cells and technical fragility of the sections on the copper grids. Transversal
and parallel (to the length of cells) sections, permitting the
same TEM details, showed the homogeneity of the layers,
especially the homogeneous density and orientation of the
B layer fibrils and of the polylamellae of A1 layer, enabling
a three-dimensional reconstruction (Fig. 32).
A combination of percentages and absolute values of the
different layers (Fig. 33; Tables 1 and 2) were found to
identify the four types of cells (upper and lower ordinary
epidermal cell cuticles, subsidiary and guard cell cuticles
belonging to stomatal apparatus). These detailed characters
provide new criteria that can be used for identification.
Considering only the percentages of thicknesses of each
layer, the upper cuticle represented by one type (30.5% of
A, 69.5% of B layer) is different from the lower one represented by three types (thinner A layer between 1.6 and
7%, thicker B layer between 93 and 98.4%). Details of the
A layer (considered as 100%) enable the same kinds of distinction: the upper cuticle has a 39% A1 layer and a 61%
A2 layer, while the three lower cuticles have a thinner A1
(between 21 and 28%) and a thicker A2 layer (between 72
and 79%).
Popa (2000) noted that one of the reasons for taxonomic
problems in pteridosperms is the great macromorphological
variability. In P. gradinarui, the minimum and maximum
values of absolute ultrastructural thicknesses are usually relatively large leading to a high variance (Table 1, called var)
and standard deviation (Table 1, called S.D.). However, measuring the variability of each layer in each cuticle type is
valuable and illustrates the necessity of a sufficient number of measurements. In addition, Popa (2000) also noted
the “unclear setting of character assessments” for this taxon.
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G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
Fig. 32. Three-dimensional reconstruction of the cuticle. OEC: ordinary epidermal cell; SC: subsidiary cell cuticle; GC: guard cell cuticle; AW: anticlinal
wall between two cells cuticle; CW: cell wall residues.
√
With the confidence interval value (= x̄ ± (var/n) × 1.96,
giving 95% ␣ risk) calculated with 30 measurements among
seven characters, a combination of affinities and disaffinities distinguishes among the four types of cuticles (Fig. 33;
Table 2) making a positive answer to Popa’s taxonomic remark, and these ultrastructural measurements could be applied to try to resolve taxonomic problems. Using only these
discriminative values of the confidence interval, a dichotomous key using the seven ultrastructural measured characters is proposed enabling identification of each type of cell
(Table 3). As eventual compression of the total cuticle can-
not be neglected during these millions of years in the sediment but seems to be impossible to check with only extinct
plants (Guignard and Zhou, in press; concerning a study on
extinct and living gingkos), the percentages of thickness of
the layers are also indicated for each type of cuticle. Ordinary epidermal cell cuticles (12.3–13.7 ␮m in mean) have
the same range of measurements, and they are separated
from stomatal apparatus cuticles (4–5.3 ␮m in mean) in total thickness and in the thickness of the B cuticular layer
(9.5–12.1 ␮m in mean versus 3.7–5.1 ␮m in mean). The tendency for stomatal apparatus cuticle to be different in thick-
Table 3
Dichotomous key enabling the identification of each of the four types of cuticles observed in P. gradinarui, using only the distinctions with the confidence
√
interval (= x̄ ± (var/n) × 1.96, giving 95% ␣ risk) among seven ultrastructural characters noted with their mean values
Thick total thickness 12.3–13.7 ␮m,
B cuticular layer 9.5–12.1 ␮m
Thin total thickness 4–5.30 ␮m, B
cuticular layer 3.7–5.1 ␮m
→
→
Ordinary epidermal
cell cuticle
Stomatal apparatus
cell cuticle
Absence of polylamellae in the A1 layer.
Cuticle proper A 4.2 ␮m, composed of
A1 layer 1.7 ␮m and A2 layer 2.5 ␮m
Presence of polylamellae in the A1 layer.
Cuticle proper A 0.2 ␮m, composed of
A1 layer 0.05 ␮m and A2 layer 0.15 ␮m
Presence of polylamellae in the A1 layer
(0.05 ␮m)
Presence of polylamellae in the A1
layer (0.08 ␮m)
→
Upper cuticle: 30.5% A;
69.5% B
→
Lower cuticle: 4.3% A;
95.7% B
→
Subsidiary cell lower cuticle:
1.6% A; 98.4% B
Guard cell lower cuticle:
7% A; 93% B
→
G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
269
√
Fig. 33. Mean and confidence interval CI (= x̄ ± (var/n) × 1.96, giving 95% ␣ risk) for each of the four types of cell cuticles. Values represent the
mean ± CI. As cuticle proper A, divided in A1 polylamellate and A2 granulous layers, shows differences hardly discernable between the three lower
cell cuticles compared with the upper ordinary epidermal cell cuticle, it is presented with two graphs: one on the left side with the four types of cell
cuticles, including upper and lower cuticles, and one with only three lower types of cell cuticles.
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G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
ness and in qualitative aspect from normal epidermal cell
cuticles has been observed before (or at least it can be seen
in the figures provided by the different following authors) in
fossil plants from various groups (belonging to Cheirolepidiaceae in Guignard et al., 1998; belonging to Conifers and
possibly related to the Cheirolepidiaceae in Archangelsky
and Taylor, 1986; belonging to Cycads or Pteridosperms
in Archangelsky et al., 1986; belonging to Bennettitales in
Villar de Seoane, 2001; belonging to Gingkoales in Villar
de Seoane, 1997). Although these differences cannot be related with the functions of stomatal apparatus as they are
fossil plants and though experiment is impossible, it can
be noted that the same kinds of differences are common in
living plants where stomata are known to be submitted to
variations in volume, opening, and closuring processes being due to water and other molecules exchanges (Larcher,
1995; Willmer and Fricker, 1996). In the case of P. gradinarui, details of cuticle proper A provide even more precise
distinctions between the four types: the upper cuticle is the
most distinct with three characters/5 left, i.e. thickness of A
cuticle proper, A1–A2 layers. Moreover as the two last characters (opaque and translucent lamellae) are missing in this
type of cuticle because of the special A1 layer (Fig. 3), it is
distinguished from the lower ordinary epidermal cell cuticle
in the dichotomous key. In the set of the three lower cuticles
remaining, subsidiary and guard cell cuticles have one distinct character (thickness of A1 layer) but it is interesting to
note that three less distinctive characters are also present yet
not taken into account in the dichotomous key (thicknesses
of: A cuticle proper, opaque lamellae in A1 layer, B cuticular layer; all indicated with // in Table 2; see also diagrams
in Fig. 33 showing a weak attachment between subsidiary
and guard cell cuticles).
4.2. Taxonomic considerations
4.2.1. Comparisons with previous studies in P. indica, P.
papillosa, P. desmomera, P. bagualensis. Time and space
considerations, technical procedures, and diagenetic
processes
Previously studied taxa show ultrastructure similar to
the present material. This is surprising since they were
collected from various regions from all over the world
distribution of this genus (Baldoni and Barale, 1996), with
different types of sediments and diagenetic histories, and
represent various time intervals. Concerning qualitative description of the cuticles, Pachypteris indica Oldham and
Morris emend. Bose and Roy (1968) (Plate I, 1 and 2 in
Bajpai and Maheshwari, 2000) was collected from the Early
Cretaceous, Sivaganaga Formation, Naicoloma India, in
arenaceous sediments. It has a thick B layer, which is their
“inner electron-lucent zone, irregularly reticulate to fibrillate
. . . .” It resembles P. gradinarui (collected from Lower Jurassic at Cristian, Brasov County, Romania, in fine sandstones)
with fibrils showing very diverse schemes. Even herring
bone fibrils are observed in their photo 2 on the right bottom
side. There are a few differences in the cuticle proper A;
they seem to describe only an A2 layer (their “outer electron
dense zone with homogeneous matrix”), indicating that it
could be an upper cuticle. But the provided magnification is
not great enough to determine the presence or absence of the
lightly stained A1 layer (see the present Fig. 2 with P. gradinarui where it is hardly visible at 1800× magnification, yet
visible at 14,500× magnification in Fig. 3). However there
is another possibility for its absence in their study, the A1
layer was not present in a few sections observed in P. gradinarui, as already noted in results. Labe and Barale (1996)
observed P. papillosa (Thomas and Bose) Harris (Middle Jurassic—Bajocian-Bathonian, Hasty Bank—England,
collected in fluvio-deltaic sediments) and P. desmomera
(De Saporta) Barale (from Upper Jurassic—Kimmeridgian,
Creys Jura—France, in lithographic limestone), and similar
features exist when compared to P. gradinarui. The upper
and lower cuticles descriptions clearly show differences between upper and lower cuticles, as P. gradinarui. They have
an A1 polylamellate layer clearly observed for P. papillosa
only in the lower cuticle as our material, however, existing
for P. desmomera in the upper cuticle, but their photo pl. II 2
resembles P. gradinarui with the special A1 layer observed
in the upper cuticle. They have also an A2 amorphous layer,
and a B fibrilous layer that is variable in ultrastructure and
described as reticulate and alveolate by the authors and
with herring bone fibrils in the case of P. desmomera (Plate
III 5). Baldoni and Barale (1996) observed P. bagualensis
(Menéndez) Baldoni and Barale (Middle Jurassic, Neuquén
Province, Argentina, collected in black clays), ultrastructure of the cuticle layers is more difficult to interpret since
the authors describe it as in poor preservation (“degradacion parcial” in their text; their Plate II 7, 8 and 9). Their
term “amorphous” could correspond to a certain level of
degradation but it is not very clear, the cuticle has an outer
dark zone without polylamellae, a middle bright zone and
an inner darker zone. In addition it can be noted that despite three different procedures (maceration and preparation
of resin blocks) used in the three Pachypteris papers, the
images reveal the same kinds of details. Nevertheless the
use of uranyl acetate-lead citrate (the present study) versus potassium permanganate in the staining (Bajpai and
Maheshwari, 2000) appears to produce clearer images.
As detailed above, precise quantitative measurements provide useful characters for identification of P. gradinarui.
Compared with shorter measurements of previous studies,
the total cuticular thickenesses of these Pachypteris species
are within the range of variation found in P. gradinarui. The
epidermal cell cuticle in P. indica (Bajpai and Maheshwari,
2000) is 10–17 ␮m thick. P. papillosa (Labe and Barale,
1996) is 22 ␮m thick for the upper epidermal cell cuticle, and
19 ␮m for the lower one. P. desmomera (Labe and Barale,
1996) is 27 ␮m in thickness for the upper epidermal cell cuticle, and 19 ␮m for the lower one. P. bagualensis (Baldoni
and Barale, 1996) has the most detailed measurements: for a
total of 1.7–2.35 ␮m the upper cuticle is 0.05–0.2 ␮m thick
G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
for their outer dark zone (2.9–8.5% of the total that could
correspond to A1 layer with degraded absent polylamellae),
0.5–0.95 ␮m for the middle bright zone (29.4–40.4% of the
total that could be the A2 granular), and 1.15–1.2 ␮m for
the inner darker zone (51.1–67.7% of the total that could be
the B layer). The lower cuticle has only one measurement
reported for the inner darker zone, which is 0.4–1 ␮m thick.
Labe and Barale (1996) indicated a thicker upper cuticle
and a thinner lower one for P. papillosa. Other pteridosperm
studies indicate this variability (Archangelsky et al., 1986;
with Ticoa harrisii, noting that “the layering of the cuticle
membrane is not uniform in the fossils and differs on the upper and lower epidermis”). However, future studies should
provide confidence intervals with their measurements of different types of cells, as they may show difference as in the
present study (Fig. 33; Table 2). In any case, they would enable comparisons with the present P. gradinarui study and
precise attribution of ultrastructural characters to a rank of
taxonomy, i.e. for instance if one character is in common
within two species its significance moves up automatically
at a higher taxonomical level (genus or family).
The relationship between fossil and living plant cuticles
is observed in rare cases of taxa containing both extinct and
extant plants (e.g. in gingkos; Guignard and Zhou, in press)
and both qualitative (description of the layers) and quantitative (measurements) data are provided. Nevertheless, the
six types of cuticles defined by Holloway (1982) are used
for fossil plant cuticles. For living plants this number is evidently very small compared with the few tens of very different genera and species of angiosperms and gymnosperms
Holloway (1982) compared. Moreover, concluded that “each
species must be considered individually and it should not be
assumed that any structural features which may be observed
are of universal occurrence” (Holloway, 1982, p. 28). This
is also true for fossil plants, although the six types of cuticles are usually found, more precise distinctions have to
be made. Even if they are successfully attributed to one of
Holloway’s six types, the features may not in fact be equivalent. For instance, in Pachypteris taxa previously studied, the
terms “close to” (“se rapprocherait de” in Labe and Barale,
1996), “approximately” (“aproxima” in Baldoni and Barale,
1996) and “close resemblance” (Bajpai and Maheshwari,
2000) are used. This is in accordance with Holloway’s observations on living plants. P. papillosa (Labe and Barale,
1996) was attributed to Holloway’s type 2 (“polylamellate
outer region sharply delineated against inner, mainly reticulate region”) and P. desmomera (Labe and Barale, 1996)
to type 1 (“outer region faintly lamellate, gradually merging
with inner mainly reticulate region”). If the P. gradinarui results have only partial affinities with Holloway’s types, the
situation becomes even more complicated as there are two
types of affinities for this single taxon (mainly type 1 for
lower cuticle, mainly type 2 for upper cuticle). For reasons
discussed above, the attribution of P. bagualensis (Baldoni
and Barale, 1996) to type 6 (“mainly amorphous”), possibly due to partial degradation, and of P. indica (Bajpai and
271
Maheshwari, 2000) to type 3 (“outer region amorphous, inner region mainly reticulate”), due to the possible absence
of outermost polylamellae, may not provide useful criteria.
4.2.2. Position within Corystospermales
According to Popa (2000), pteridosperm taxa represent
a morphologically diverse and still not well understood
group, especially when examining leaf form genera without
attached reproductive structures. In the Corystospermales,
the Northern Hemisphere, European genera, such as
Pachypteris (including here the junior synonym Thinnfeldia), Cycadopteris, Komlopteris, and Rhaphidopteris, have
some differences with other corystosperm form genera, such
as the Southern Hemisphere Dicroidium. Only Dicroidium
and Pachypteris are known as true corystosperms by their
reproductive organs. The other genera, as Komlopteris,
Rhaphidopteris and Cycadopteris, are reported to corystosperms by their vegetative affinities. For this reason, they
may be not true corystosperms, and it can explain their ultrastructural differences. Corystosperms may not represent
a natural taxon.
4.2.2.1. P. gradinarui and the genus Cycadopteris. Cycadopteris brauniana Zigno emend. Barale (Labe and
Barale, 1996), the only species where ultrastructure has been
examined within this genus, has two different types of cuticles (upper and lower, but less distinct than for Pachypteris
species reported in the same article) as most Pachypteris
species described above. However, other similarities seem
to be absent. The upper cuticle is striate and rarely lamellate
(Fig. 1A and B, Labe and Barale, 1996), above an amorphous and inner electron lucent zone. The lower cuticle is
sparsely lamellate, followed by an amorphous zone and an
innermost alveolate layer. The lower cuticle (7 ␮m) is included in our measurements, the thickness of the upper cuticle (40 ␮m) is not observed in P. gradinarui (maximum of
23.63 ␮m). When compared to C. brauniana, P. gradinarui
has more ultrastructural similarities with other Pachypteris
than with Cycadopteris. Popa (2000) recognized macromorphological affinities between the two genera Pachypteris
and Cycadopteris, and suggested that P. gradinarui may be
a transitional taxon between these two genera. However, the
ultrastructure of the cuticle does not support this interpretation. Based on these ultrastructural features, Pachypteris
and Cycadopteris may not be that related. It was also proposed with the macromorphological study of Barale (1982).
As with Pachypteris, more detailed ultrastructural study
may be useful for taxonomic resolution within Cycadopteris
too.
4.2.2.2. P. gradinarui and the genus Komlopteris. Cuticular ultrastructure of Komlopteris is very different from
Pachypteris and Cycadopteris. Moreover, within Komlopteris many features are in closely related taxa, and thus
perhaps these characters can be used at a higher taxonomic level than species. Maheshwari and Bajpai (1996a),
272
G. Guignard et al. / Tissue & Cell 36 (2004) 263–273
Bajpai (1997) and Bajpai and Maheshwari (2000) described
the cuticle of Thinnfeldia indica Feistmantel (Komlopteris
indica (Feistmantel) Barbacka; Barbacka, 1994). Bajpai
and Maheshwari (2000) discussed the differences between
Komlopteris indica and Pachypteris indica. Despite fungi
and technical problems (electron dense spots), the ultrastructural similarities between Komlopteris indica and K.
nordenskioeldii (Nathorst) Barbacka (studied in Guignard
et al., 2001; see also Barbacka, 1994; Barbacka and van
Konijnenburg-van Cittert, 1998) are striking. These cuticles
are amorphous, and consist of zones with variable granule
densities (Figs. 6 and 7, Bajpai, 1997; Figs. 1–3, Maheswari
and Bajpai, 1996a; Plate I 3, Bajpai and Maheshwari, 2000;
see also the reconstruction of the cuticle Fig. 1, Guignard
et al., 2001). The cuticle is constituted of only cuticle proper
A (only A2, with an absence of A1 outermost layer), and
thus is very different from the Pachypteris gradinarui cuticle, except for the pillar shape structure above the anticlinal
walls (Guignard et al., 2001, compare Fig. 4 and their Plate
V 2 in shade cuticles).
measurements of the features distinguish upper and
lower epidermal and stomatal cell types.
(2) Three-dimensional reconstruction and a dichotomous
key (Table 3) using a combination of ultrastructural characters illustrate a difference between the four types of
cell cuticles: ordinary epidermal cell cuticles of both
upper and lower cuticles, and subsidiary and guard cell
cuticles belonging to stomatal apparatus.
(3) Comparisons with other ultrastructural studies of
Pachypteris species seem to show that diagenetic processes, time and space, and/or processes related to
technical procedures, are minimal within this genus.
(4) This study provides a new approach that may be useful
for the comparison of the cuticular ultrastructure among
fossil plants. Further comparisons with other species of
the same genus could enable to state about the importance of characters as criteria at species or at different
taxonomic ranks. These characters are: total thickness of
the cuticle, A cuticle proper and B cuticular layer thickness; A1 and A2 layers thickness; presence or absence
of polylamellae in the A1 layer and their thickness.
4.3. P. gradinarui and the genera
Rhaphidopteris—Dicroidium
Acknowledgements
The cuticular ultrastructure of Pachypteris gradinarui
varies from these two other taxa, supporting the macromorphological differences noted by Popa (2000). Resemblances
also exist between Rhaphidopteris and Dicroidium but are
less similar than Komlopteris comparisons. Rhaphidopteris
fragilis Barale (Barale, 1972) has a complex B layer composed of different zones (see Fig. 4A in Labe and Barale, 1996). The polylamellate layer has regularly arranged
lamellae in the upper part and sparse, irregularly arranged,
translucent lamellae in the lower part (see their Plate IV
6–9). In this respect the cuticle has ultrastructural affinities
with Gingkoales cuticles (Guignard and Zhou, in press).
Moreover, recent studies have shown that Rhaphidopteris
is heterogeneous (Zhou and Zhang, 2000; Zhou et al.,
2001), and some of the species are closely comparable
with ginkgoalean fossils in gross morphology and cuticular
structure. Even the lysigenous resin bodies characteristic
of ginkgos have been found in the leaves of one species,
R. cornuta Zhang and Zhou (1996). Maheshwari and
Bajpai (1996b) have studied Dicroidium gouldii Retallack
(Retallack, 1977), and show some affinities with Rhaphidopteris. For example D. gouldii also has a complex B
layer (see their general views at low magnification in Plate
12 and Plate 21 for both cuticles of pinnules). Maheshwari
and Bajpai (1996b) also described a Dicroidium sp., but
however they were uncertain about placing it in this genus.
5. Conclusions
(1) Quantitative descriptions of the cuticular ultrastructure of P. gradinarui were developed and statistical
This research was partly supported by Université
Claude-Bernard Lyon 1, with the Professorship offered to
Dr. Mihai E. Popa during the summer of 2002. The cuticle
material was kindly provided by Dr Eugen Gradinaru. We
wish to thank M. Nicolas Labert for the technical assistance and members of the Centre technologique des microstructures CTM, Université Claude-Bernard 1, France.
Many thanks are due to Dr. Lisa Boucher and Dr Jennifer
McElwain for their English linguistic corrections.
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