Cour. Forsch.-Inst. Senckenberg | 258 | 39 – 55 |
4 Figs, 1 Tab.
| Frankfurt a. M., 15. 11. 2007
The Cuticle Database:
Developing an interactive tool for taxonomic and paleoenvironmental study of the fossil
cuticle record
With 4 figs, 1 tab.
Richard Barclay, Jennifer McElwain, David Dilcher & Bradley Sageman
Abstract
Fossil cuticle usually preserves a perfect replica of the plant epidermis and thus provides an unparalleled
record of epidermal micromorphological characters useful for answering a variety of scientific questions.
Paleobotanical applications involving cuticle have focused either on taxonomic identification of species
or on the assessment of paleoecological and paleoenvironmental conditions. Taxonomic identification
using epidermal characters has long been an endeavor of paleobotanists. The most conservative approach
has been to assign specimens to morphotypes, while specimens with more characters have facilitated the
establishment of new species in fossil genera, usually within modern families. In certain cases, epidermal
characters have been combined with information from other plant organs to compare coeval floras from
different basins. Recent research has benefited from an improved understanding of the relationships between
the morphology preserved by cuticle and the paleoenvironmental conditions where the plant developed.
However, these studies are limited by the difficulties in the identification of fossil cuticle, in particular
dispersed cuticle, which often provides the best temporal resolution for paleoenvironmental study. To address this key limitation, we have developed an Internet-accessible database of cuticle images, referred to
as the Cuticle Database Project (or Cuticle), which will facilitate the identification of fossil cuticle material
through the development of an identification key structure. This database will help to facilitate identification
of cuticle specimens and to advance attempts to distinguish which epidermal characters are environmentally
controlled, http://fm1.fieldmuseum.org/cuticule/PaleoCollaborator
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K e y w o r d s: Cuticle Database Project, PaleoCollaborator, angiosperms, fossil, taxonomy, stomatal
frequency, stomatal distribution, Florida Museum, Field Museum
Introduction
The plant cuticular membrane is a three-dimensional matrix of wax, cutin, cellulose, and pectin. Cuticular waxes are
deposited over the epidermal cells, filling in the interstitial
spaces of cutin fibers that are adjacent to a membrane layer
of cellulose and pectin (Martin & Juniper 1970, Halloway
1971). The primary function of plant cuticle is to prevent
water loss to the external environment. This key innovation, identified in the earliest vascular plants (Edwards et
al. 1992), was instrumental in the process of terrestrialization in the Paleozoic (Edwards 1986). Although the cellulose that makes up plant cell walls degrades rapidly, the
cuticular membrane is extremely resistant to the oxidative
and reducing conditions of depositional environments,
facilitating the preservation of cuticle in the fossil record.
The high preservation potential of cuticle combined with
the fact that it usually preserves an excellent impression of
the leaf epidermis, and thus provides an excellent record of
epidermal micromorphological and anatomical traits, make
it an important paleobotanical resource for taxonomic
and ecological studies. Dispersed fragments are the most
abundant type of cuticle and are found in most terrestrial
and nearshore sedimentary facies, but macrofossil compressions also commonly preserve cuticle. Paleobotanical
studies of cuticle began in the mid 1800s with the work of
Brodie (1842) who examined cuticle from clay deposits
Authors’ addresses: Richard Barclay (corresponding author) & Bradley Sageman, Northwestern University, Department of Earth and Planetary
Sciences, 1850 Campus Drive, Evanston, IL 60208, USA, <[email protected]>, <[email protected]>; Jennifer McElwain, University College Dublin, School of Biological and Environmental Science, Belfield Dublin 4, Ireland, <[email protected]>;
David L. Dilcher, Florida Museum of Natural History, Paleobotany and Palynology Laboratory, Gainesville, FL 32611-7800, USA, <dilcher@
flmnh.ufl.edu>
© E. Schweizerbart’sche Verlagsbuchhandlung (Nägele u. Obermiller), 2007, ISSN 0341–4116
Barclay et al.: The Cuticle Database: Developing an interactive tool for study of the fossil cuticle record
eschweizerbartxxx sng-
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Cour. Forsch.-Inst. Senckenberg, 258, 2007
on the English Hampshire coast. Subsequent researchers
have studied fossil cuticle because it preserves a variety of
epidermal traits applicable to a number of scientific questions. Many studies have utilized cuticle to constrain the
taxonomic affinity of fossil plants or to determine paleoenvironmental conditions including paleoclimate and atmospheric carbon dioxide (CO2) concentration (Beerling &
Chaloner 1992, McElwain & Chaloner 1995, Kürschner
et al. 1996, Royer 2001). Where macrofossil assemblages
are not preserved, the dispersed cuticle record provides
another means of estimating the species richness of ancient
plant communities, and to track plant composition through
time or across different depositional environments (Tu et
al. 1999, 2002, Kovar-Eder et al. 2001).
Before it is feasible to address research questions
that use the abundant fossil cuticle record, it is important
to distinguish between epidermal characters reliable for
taxonomic and systematic purposes (Dilcher & Zenk
1968) and those with greater utility for paleoenvironmental reconstructions. Taxonomic studies require characters
that are anatomically conservative and largely unaffected
by environmental variables. For paleoatmospheric and
paleoenvironmental reconstructions we require anatomical characters that can be calibrated to changes in known
environmental parameters. In both types of study, fossil
cuticle needs to be compared with a large dataset of modern cuticle, spanning as wide a taxonomic range as possible, in order to reliably constrain the taxonomic affinity
of the fossil cuticle in question, or to infer the paleoenvironmental conditions in which the plant developed.
To facilitate this comparison we need a means of
rapidly sorting through an enormous set of cuticles from
modern and fossil plant groups. As is common in paleontological research, individual collections tend to be
widely dispersed and the logistics of developing a truly
comprehensive database are daunting. The advent of the
Internet, however, has provided an effective vehicle for
cuticle researchers to rapidly collaborate on the task,
given an appropriate framework. This paper describes
the development of that framework. We are developing
an Internet accessible image database and identification key that contains images of both modern and fossil
plant cuticle that encourages data sharing and analysis
across the Internet. The Cuticle Database Project (called
Cuticle) is an attempt to resolve some of the key issues
that currently hinder taxonomic, systematic, and paleoecological investigations of dispersed cuticle in the plant
fossil record (fig. 1). In this paper we briefly review
recent paleobotanical literature on fossil cuticle, focusing on the particular characters used by paleobotanists to
either identify specific fossil cuticle or to infer paleoenvironmental parameters. We provide an overview of
how Cuticle is constructed, and how it provides a means
to directly compare fossil and modern material using
equivalent epidermal characters. Lastly, we discuss how
this new online resource can be used for taxonomic and
paleoenvironmental study of the fossil cuticle record.
Angiosperm epidermal characters
preserved in fossil cuticle
In the following review of epidermal character usage
in paleobotanical research the discussion is focused on
angiosperms and the literature produced during the past
several decades. Two previous reviews have broadly covered the history of cuticle use in paleobotany from 1842
to 1995 (Dilcher 1974, Upchurch 1995). The discussion
here will focus first on plant epidermal characters with the
greatest utility for taxonomic analysis, and then on key
epidermal characters used to reconstruct paleoclimatic
and paleoenvironmental conditions. Different aspects of
these two topics have been reviewed elsewhere (Beerling
& Chaloner 1993, van der Burgh et al. 1993, Kürschner
et al. 1996, McElwain & Chaloner 1996, Wagner et al.
1996, 1999, 2000, 2004, Kürschner 1997, Royer 2001,
Royer et al. 2001, van Hoof et al. 2005).
Taxonomic Identification Using Plant Cuticle
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The Utility Of The Stomatal Complex
One of the most conspicuous features of the epidermis
that is preserved in leaf cuticle is the stomatal complex.
Stomata comprise two elongated guard cells bracketing a
stomatal pore, and often, but not always, are surrounded
by one to many subsidiary cells. Subsidiary cells are
derived from a stomatal initial cell, as opposed to an epidermal pavement initial cell (Metcalfe & Chalk 1950,
van Cotthem 1970, Croxdale et al. 1992). Subsidiary
cells can form a conspicuous arrangement around the
guard cells, and the terminology used to describe these
arrangements has been discussed for over a century. For
a detailed review of the historical progression of stomatal
complex terminology see Baranova (1987, 1992) and
Prabhakar (2004).
Debate over the appropriate set of terms to describe
stomatal complexes continues (Prabhakar 2004). It was
shown at the outset of cuticular investigation that the development of the stomatal complex follows a predictable
ontogenetic pathway towards a mature stomatal arrange-
Fig. 1: A) Cuticle homepage: http://fm1.fieldmuseum.org/cuticle/PaleoCollaborator. B) Final page of Cuticle identification key. C)
Search page thumbnails for a search of all 269 species of Lauraceae. D) Detail page of Ocotea cudadontissi (Lauraceae), FLMNH
#253.
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Barclay et al.: The Cuticle Database: Developing an interactive tool for study of the fossil cuticle record
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Fig. 2: Stomatal complex types. Reference in left column is original source of name. Images of extant species from the Florida
Museum collection, modified from Baranova (1987, 1992) & Yaojia et al. (1999). Drawings and definitions modified from Dilcher
(1974).
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Cour. Forsch.-Inst. Senckenberg, 258, 2007
ment (Borodin 1888, Vesque 1889). If these developmental paths could be defined and then shown to be uniquely
associated with particular taxonomic groups, taxonomists
would have had an additional and powerful tool to help
distinguish natural plant groups. Time has proven this to
be a complicated endeavor. Vesque (1889) outlined six
ontogenetic pathways, four of which produce distinguishable mature stomatal complexes. Most of the subsequent
work aimed at describing stomatal complexes of modern
plant families focused only on the mature state of the
stomatal complex rather than their ontogenetic pathways.
Gradually the ontogenetic terms used by Vesque (1889),
such as ranunculaceous, became synonymous with the
mature stomatal complex arrangement.
This practice led to widespread confusion because
different ontogenetic pathways could produce the same
mature stomatal complex type. The terms for stomatal
complex types had been created by adopting a modified
family name from where they were originally discovered
(e.g. ranunculaceous for the Ranunculaceae; fig. 2), but
it became apparent that in many cases different stomatal
complex types were rarely exclusive to just one plant family (Baranova 1987). Also, the overwhelming majority of
fossils exhibit only the mature stomatal arrangement, so
paleobotanists did not frequently apply Vesque’s scheme.
This is mostly because the availability of fossilized developmental series is a very rare occurrence, but there
are a few notable exceptions (Barbaka & Boka 2000).
Several reviews are available for ontogenetic classification
schemes (Fryns-Claessens & van Cotthem 1973, Stevens
& Martin 1978, Payne 1979, Timonin 1995).
New terms were needed to refer to the stomatal complex type that were based purely upon the mature arrangement of cells surrounding the stoma, independent
of ontogeny and taxonomy (Metcalfe & Chalk 1950).
The four distinguishable mature types of Vesque (1889)
are ranunculaceous, cruciferous, caryophyllaceous, and
rubiaceous (figs 2 and 3). These were renamed by Metcalfe & C halk (1950) with new terms: anomocytic,
anisocytic, diacytic, and paracytic respectively (figs 2 and
3). The next forty years of research by plant anatomists
produced ten more stomatal complex types (Stromberg
1956, Metcalfe 1961, Stace 1965, van Cotthem 1970,
Fryns-Claessens & van Cotthem 1973, den Hartog &
Baas 1978, Baranova 1983, Baranova 1987, Wilkinson
1989). Baranova (1987, 1992) determined that fourteen
stomatal complex types could encompass the myriad
terms previously used in the literature: actinocytic, anisocytic, anomocytic, desmocytic, diacytic, encyclocytic,
hemiparacytic, laterocytic, paracytic, pericytic, polocytic,
staurocytic, stephanocytic, and tetracytic (figs 2 and 3).
While these mature classification types are conceptually useful, at times their practical taxonomic value can be
limited (Baranova 1987) because more than one stomatal
complex type may occur in the same genus or species, or
even on the same leaf surface (Metcalfe & Chalk 1950,
den Hartog & Baas 1978, Baranova 1987, Carpenter
2005). A recent investigation of Amborella, Nymphaeales,
Schizandraceae, Trimeniaceae, and Austrobailea showed
that multiple stomatal complex types are present even in
these basal angiosperm clades (Carpenter 2005). The ancestral condition is typically simple, grading between anomocytic (no true subsidiary cells, fig. 2) and stephanocytic
(subsidiary cells loosely arranged in a rosette, fig. 2), with
more complicated stomatal complex types forming in more
derived seed plant lineages (Carpenter 2005). It is also common for genera in the same family to have different types of
stomatal complexes (Baranova 1987). For example, in the
Chloranthaceae, Ascarina has encyclocytic stomata (fig. 2)
yet closely related Hedyosmum has stephanocytic stomata
(fig. 3, Baranova 1987). A survey of the taxonomic position
of the laterocytic (fig. 3) stomatal type found this configuration to be present in fourteen different, largely unrelated
families (figs 2 and 3, Baranova 1983).
With only fourteen distinct mature stomatal types (as
well as their subtypes) recognized among angiosperms,
the stomatal complex alone cannot help to resolve the
453 different extant angiosperm families (Judd et al.
2002, Soltis et al. 2005), or definitively constrain the
identity of fossil cuticle to a specific taxonomic rank.
Other epidermal characters, such as presence/absence
of trichomes, trichome type, epidermal physiogonomy,
and micromorphology are therefore required in conjunction with stomatal complex type in order to determine
taxonomic affinity of fossil plant cuticle. We review the
utility of these additional characters for taxonomic and
ecological study in later sections.
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Approaches to Taxon Identification
Using suites of epidermal characters, including but not
limited to, trichome type and arrangement, presence or
absence of papillae, in addition to stomatal complex (tab.
1), provides a more robust approach for constraining the
taxonomy of modern and fossil cuticle specimens (tab. 1).
Researchers have taken three primary approaches to the
identification of dispersed cuticles with regard to placing
them in a phylogenetic context: 1) cuticles are assigned to
morphotypes; 2) they are binned into broad form genera;
or 3) described as new fossil genera or species. The most
cautious approach has been to treat the specimens strictly
as morphotypes (Harvey 1978), making little or no attempt to determine the taxonomic identity of a specimen.
When taxonomic identity is not the focus, morphotypes
can be of great utility, such as when attempting to establish Eocene biozones using very large datasets of dispersed cuticle (Rowett & Sparrow 1994). With well-preserved Eocene material, epidermal characters have been
used to establish form genera based upon an artificial
classification system (Roselt & Schneider 1969, Kovach
& Dilcher 1984). The mature stomatal complex type and
their arrangement on the leaf surface were the primary
43
Barclay et al.: The Cuticle Database: Developing an interactive tool for study of the fossil cuticle record
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Fig. 3: Stomatal complex types continued. Reference is original source of name. Images of extant species from the Florida Museum,
and modified from Baranova (1987, 1992) & Yaojia et al. (1999). Drawings modified from Dilcher (1974). Definitions from
Dilcher (1974) and Baranova (1987).
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Cour. Forsch.-Inst. Senckenberg, 258, 2007
LEAF
CUTICLE
Table 1: A comparison of cuticle and leaf characters controlled predominantly by genetics (genotype) versus the environment
(phenotype).
Major Characters
Papillae type and position
Secretory structures (glands, glandular hairs)
Stomatal complex type
Stomatal distribution (rows, dispersed, sunken)
Stomatal position (amphistomatous, hypostomatous)
Striations (intercellular, intracellular, epicuticular wax)
Trichomes (type, complexity, location)
Trichome bases plus surrounding cell arrangement and number
Cell walls (degree of crenulation)
Hydathodes (position, size, number)
Papilla number and size
Stomatal density
Stomatal index
Trichome density
Palisade parenchyma (structure)
Palisade to mesophyll parenchyma ratio
characters used to create form genera in this example
(Roselt & Schneider 1969). Kovach & Dilcher (1984)
identified 29 distinct morphospecies in their dispersed
cuticle assemblage from the middle Eocene Claiborne
Group of Tennessee, USA. These authors recognized that
several of the species were likely of Lauraceae affinity,
but refrained from making any taxonomic assignments
without more definitive characters, especially from other
plant organs.
When more information is available, the establishment
of new fossil genera and species is justifiable. Often, the
available characters are only sufficient to suggest affinity
to modern families and orders (Upchurch & Dilcher 1990,
Upchurch 1995). Taxonomic determination of monocotyledons has received some attention, such as studies of
Campanian monocots from Austria (Kvaček & Herman
2004), cuticle-bearing macrofossils in the Araceae from
the European Tertiary (Wilde et al. 2005), and middle
Miocene grasses from Kenya (Dugas & Retallack 1993).
Dicotyledons have been the predominant focus of angiosperm cuticle studies, starting early in the 20th century with
the work of Bandulska, who placed Eocene fossil cuticle
in Fagaceae (Bandulska 1924) and Lauraceae (Bandulska
1926). This original work has been continued by careful
investigations of North American Eocene fossils (Jones &
Dilcher 1980, Herendeen & Dilcher 1990), Eocene fossils from Australia (Christophel & Lys 1986, Hill 1986),
and Cretaceous fossils (Albian and Campanian) from the
Western Interior of the United States (Wolfe & Upchurch
1987, Upchurch 1995).
The most comprehensive approach has been to identify all of the species in a flora using both epidermal
characters preserved in fossil cuticle in conjunction with
macromorphological characters of the leaf (Kovar-Eder
et al. 1998). Fossil species identified in this manner are
Genotype
X
X
X
X
X
X
X
X
Phenotype
X
X
X
X
X
X
X
X
X
X
X
then compared with those of other coeval floras from
different regions (Kovar-Eder et al. 2001). This type of
study provides important evolutionary and ecological
information and can best be undertaken with floras that
are Miocene or younger, simply because less evolutionary divergence has occurred, and fossils are more easily
related to modern groups.
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Paleoecological Evaluation Using Plant Cuticle
Paleoatmospheric and paleoenvironmental reconstructions require anatomical characters with a high degree
of ecophenotypic plasticity, so characters are needed
that are predominantly controlled by subtle differences
in environmental parameters such as temperature, water
availability, light intensity, and atmospheric composition.
Botanists have identified epidermal characters with this
high degree of ecophenotypic variability, and rigorous investigation of the degree to which these characters can be
modified by environmental parameters has led to entirely
new avenues of paleobotanical research over the past
two decades (Woodward 1987, Beerling & Chaloner
1992, McElwain & Chaloner 1995, Kürschner et al.
1996, McElwain et al. 1999, Wagner et al. 1999, Royer
2001, Royer et al. 2001). Studies of paleoecology and
paleoclimates have been greatly enhanced by the use of
fossil plant cuticle and the anatomical detail it preserves.
However, the relationships between plant anatomy and
environmental conditions are often species-specific. So
the fact that it has been difficult to determine if fossil
cuticle specimens are assignable to the species level, or
are only diagnostic to the level of genus or family has
hindered efforts to calibrate anatomical features with
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Barclay et al.: The Cuticle Database: Developing an interactive tool for study of the fossil cuticle record
specific environmental parameters. This is why it is imperative that comparative databases such as Cuticle are
available to researchers globally. Identifying the species
of plants in an ecosystem is intricately connected to a
greater understanding of how these same plants respond
to environmental changes. The need to separate characters that are primarily controlled by genetics from those
controlled by environmental conditions will become
increasingly important.
Paleoecology
Within paleoecology, recent investigations have focused
on reconstructing forest canopies (Kürschner et al. 1996,
Arens 1997, Chen et al. 2001, Denk & Velitzelos 2002),
determining plant habit (Krings et al. 2003), as well as
water availability (Tu et al. 1999, 2002, Haworth et al.
2005). This is possible because leaf morphology and
anatomy respond strongly to the large gradients in light
intensity, temperature, humidity, and CO2 partial pressure
present within forest canopies. Many of these responses
occur at the leaf surface, in the epidermis, and thus are
recorded in fossil leaf cuticle (Givnish 1986 & references
therein). As a result, there are a number of prominent
epidermal characters preserved in the fossil cuticle record
that have great utility for paleoecological interpretation.
One product of this environmental gradient is that sunleaves have different morphological characteristics than
shade-leaves (Arens 1997, Kürschner 1997). The degree
of crenulation to the anticlinal cell walls can be used to
differentiate sun versus shade leaves in modern and fossil
plants (Kürschner 1997). If the internal anatomy of fossil
leaves is preserved, then the thickness of the epidermis
and hypodermis, the ratio of palisade to spongy mesophyll, and the structure of palisade parenchyma can be
used to infer light intensity as these are phenotypically
plastic (Arens 1997, Kürschner 1997). These leaf characters, as well as other plant features, were used to refine
ecological interpretations of the Pennsylvanian aged
medullosan pteridosperms Alethopteris and Neuropteris
(Arens 1997).
The presence of papillae on the surface of epidermal
cells of modern rainforest understory herbaceous plants
has been shown to enhance the harvest of light for photosynthesis in deeply shaded sub-canopy environments
(Haberlandt 1914, Lee 1986, Lee et al. 1990). Papillae
are thought to function as lenses concentrating light into
the chloroplasts, which tend to be localized under the
cone of light in these light-limited conditions (Bone et
al. 1985, Lee et al. 1990). This functional interpretation
of epidermal papillae is in marked contrast to the more
traditional view among paleobotanists that papillae are
aridity or stress indicators, serving to minimize water loss
in dry/harsh environments by increasing the boundary
layer resistance on the leaf surface (Watson 1988, Alvin
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46
et al. 1994). It is likely that papillae serve multiple functions in plants and that their original function may have
been co-opted at some point in their evolutionary history.
It is notable that many of the basal angiosperms possess
epidermal papillae and the majority of taxa in the ANITA
clade today are found in dark understory tropical environments (Feild et al. 2004). However, we still have much
to learn about how and if the basal angiosperms living in
dark understory tropical environments are related to the
ancestral angiosperms found in the fossil record.
Stomatal density (stomata per mm2 of leaf surface)
is a character usually reserved for estimating paleoatmospheric CO2 concentration (Woodward 1987, Royer
2001). However, where large datasets of fossil leaf cuticle are available for the same morphotype it can also be
used to interpret plant habit. For instance, stomatal densities tend to be lower for shade-adapted leaves growing
in reduced light intensities under the canopy (Salisbury
1927, Poole et al. 1996, Kürschner et al. 1996, Kürschner 1997, Wagner 1998, Royer 2001). The thickness of
cuticle has also been suggested as a character for determining if plants are deciduous or evergreen (Litke 1967)
since evergreen species generally have thicker cuticle
than deciduous species, particularly on the abaxial surface (Upchurch 1995). Herbaceous and woody deciduous species have cuticle less than 3μm thick, while that
of evergreen woody species is typically 4–5μm thick.
Cuticle of xeromorphic plants can be as thick as 60μm
(Ihlenfeldt & Hartmann 1982) and variation in thickness often reflects acclimation to water regimes (Turner
1994). There are several notable exceptions, however,
which suggest that cuticle thickness alone cannot always
be used to infer the leaf lifespan of fossil leaves or water
regime (McElwain & Chaloner 1996).
Atmospheric CO2
Studies of stomatal frequency on modern plant leaves
(Salisbury 1927) greatly predated the discovery that
changes to the number of stomata was inversely correlated with atmospheric CO2 (pCO2) (Woodward 1987,
Woodward & Bazzaz 1988). Both stomatal density and
stomatal index (proportion of stomata to total epidermal
cells) have been demonstrated to respond to changes in
pCO2 over time scales applicable to the fossil record. The
stomatal frequency method has been applied to the entire
Phanerozoic to estimate paleoatmospheric CO2 levels,
including the Holocene (Beerling & Chaloner 1992),
the Neogene (van der Burgh et al. 1993, Kürschner et
al. 1996), the Paleogene (McElwain 1998, Royer et al.
2001), the Mesozoic (McElwain et al. 1999, Haworth et
al. 2005, McElwain et al. 2005), as well as the Paleozoic
(McElwain & Chaloner 1995, Beerling & Woodward
1997). The relationship of stomatal frequency on fossil
cuticle to changes in paleoatmospheric composition has
Cour. Forsch.-Inst. Senckenberg, 258, 2007
been comprehensively reviewed (Beerling & Chaloner
1993, Woodward & Kelly 1995, Royer 2001).
Despite the advances in our understanding of how
atmospheric composition has changed through the Phanerozoic, there are still problems associated with this
method, particularly that of calibration because relationships between leaf stomatal frequency and atmospheric
CO2 concentration are often species-specific. Providing
accurate estimates of paleo-CO2 concentration from fossil plant stomatal frequency relies on an ability to first
assign fossil cuticle to modern families, genera, or ideally
species, for comparison when creating the calibration.
In the case of extinct taxa, fossil cuticle can be assigned
to morphotypes or fossil taxa, the stomatal frequencies
of which are then compared with suitable nearest living
morphological or ecological equivalents for calibration
(e.g. Haworth et al. 2005). This limits the power of the
stomatal-pCO2 proxy method due to the inherent difficulties associated with identifying cuticle specimens in the
fossil record, especially in the absence of macrofossils.
By developing the large comparative database, Cuticle,
we will be able to improve the speed and quality of identifications of cuticle in the fossil record, and therefore
help to more rapidly advance calibration of fossil stomatal frequency change in terms of paleoatmospheric CO2.
(calcium in halophytic plants) and secretion of harmful or
defensive compounds respectively, and in the acquisition
of water and/or nutrients in many epiphytes (see review by
Gutschick 1999). The placement of trichomes on the leaf
surface is also critical. Trichome occurrence in modern
tropical rainforest taxa is often restricted to the abaxial leaf
surface (Richards 1952). When this character is observed
in fossil assemblages, along with a high percentage of
taxa with coriaceous cuticle (4–5μm thick) and an overall
smooth surface to the cuticle, periods of high rainfall have
been inferred (Wolfe & Upchurch 1987, Upchurch 1995).
Interpretation of paleoenvironmental conditions in the fossil record using the presence/absence and/or abundance of
trichomes on fossil leaf cuticle is complex because of these
multiple potential functions.
In modern plants, water can be rapidly released by guttation through hydathodes, special structures that secrete
liquid water through pores of stomatal origin, but remain
permanently open to the external environment. They are
common in modern tropical vines and lianas as an adaptation to reduce root water pressure (Ziegenspeck 1948), and
in the Chloranthaceae have been shown to reduce flooding
of the mesophyll tissue that can reduce photosynthesis
(Feild et al. 2005). Evidence for hydathodes in fossil leaf
cuticle has been used to interpret plant synecology, such as
a vining habit for some Paleozoic pteridosperms (Krings
et al. 2003).
Paleoclimate
Isotopic Analysis
The occurrence and abundance of trichomes (leaf hairs)
and epidermal papillae on fossil leaves has commonly
been used to infer water availability in past environments,
or to identify intervals of drought from high rainfall in
stratigraphic successions. This is based on the premise that
trichome density is often high in taxa adapted to arid conditions (Coulter et al. 1930, Fahn 1982). A high density
of trichomes and/or papillae is presumed to increase the
leaf boundary layer, slowing diffusion of gases through
the stomatal pore, thereby reducing transpirational water loss. Recent ecophysiological investigation of foliar
trichome function in the epiphytic C4 plant Tillandsia,
have confirmed that they increase the leaf boundary layer,
however no significant effect on transpirational water
loss was observed (Benz & Martin 2006). Although trichomes may help to balance leaf temperatures by reflecting UV radiation (Ehleringer 1981), the results of Benz
& Martin (2006) suggest that the common interpretation
of trichomes as aridity indicators in the fossil record may
be oversimplified. For instance, an increase in the number
of taxa with trichomes on the upper surface of dispersed
fossil cuticle was used to suggest an interval of aridity
during the angiosperm recovery phase following the Cretaceous-Paleogene mass extinction boundary (Wolfe &
Upchurch 1987). This interpretation did not consider that
trichomes also function to protect against UV radiation, in
defense against herbivory (Letourneau 1997), in storage
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Stable carbon isotope analysis (δ13C, Hydrogen/Deuterium ratio) of modern and fossil plant cuticle, as well as
molecules specific to the terrestrial plant cuticle (long
chain n-alkanes), has demonstrated that changes in the
isotopic composition of the atmosphere can be detected
by parallel changes in the isotopic composition of plants
(Grocke et al. 1999, Arens et al. 2000, Hesselbo et al.
2002, Hesselbo et al. 2007). These studies have paved
the way for a better understanding of the causes and
consequences of past carbon cycle dynamics using δ13C
of fossil plant cuticle, and are increasingly used to constrain carbon-cycle dynamics and the linkages between
terrestrial and marine carbon reservoirs. Analysis of
δ13C in cuticle across the Triassic-Jurassic (McElwain
et al. 1999), and Cretaceous-Tertiary (Arens & Jahren
2000, Lomax et al. 2000) mass extinction boundaries has
provided compelling evidence for major perturbation to
the global carbon cycle coincident with mass extinction
in the marine realm. It has also been demonstrated that
records of δ13C from fossil plant material are comparable
to those from marine organic carbon during the entire
Early Cretaceous (Jahren et al. 2001) providing further
support that changes in global atmospheric δ13C values
are the primary controlling factor influencing δ13C values
in terrestrial plants.
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Barclay et al.: The Cuticle Database: Developing an interactive tool for study of the fossil cuticle record
Theoretical models predict that any change in the balance of carbon loss from (e.g. burial) and release into the
atmosphere (e.g. volcanically released CO2) will alter the
isotopic composition of the atmosphere (δ13Catm). Increased
burial of carbon is expected to result in a positive excursion in atmospheric δ13C. Analysis of the δ13C of presumed
terrestrial plant material taken from Japanese marine sections record a >2‰ (per mil) positive excursion at the
Cenomanian-Turonian boundary (Hasegawa 1997), paralleling a 2‰ positive excursion in organic carbon found
in marine sections, and providing support for theoretical
models of the carbon cycle (Pratt 1985, Arthur et al.
1988). Preliminary analyses of terrestrial plant material
from paralic facies of the Dakota Formation in western
Utah corroborate this finding (Barclay et al. 2006), where
specimens of cuticle, coal, and charcoal exhibit a 2‰
positive excursion in all three records directly at the onset
of enhanced marine organic carbon burial. Stable carbon
isotope signatures have also been utilized to determine
environmental stresses on plants such as gradients in water availability and soil salinity that affect the distribution
of plants on paleo-landscapes (Tu et al. 1999, 2002), and
more recently as a paleo-precipitation proxy (Pedentchouk
& Pagani 2005, Smith & Freeman 2006).
Cuticle Database Development
Identification Key
eschweizerbartxxx sng-
One of the main objectives of this project is to create a
means of rapidly constraining the taxonomic identity of
fossil cuticle specimens of unknown affinity in order to
address some of the issues raised in this review. Large
digital archives of cuticle images will facilitate the development of alternative methods for interpreting anatomical
changes of extinct plants in relation to environmental
change. The quality of paleoenvironmental interpretations will increasingly depend upon the accuracy of the
taxonomic identification of fossil specimens and an ability to further constrain the functional interpretation of
leaf anatomical characters. For instance, are particular
foliar trichome morphologies associated with specific
functions? Can specific papillae on fossil leaf surfaces be
correlated to dark understory ecologies or did they serve a
protective function in high light environments? Databases
such as Cuticle will help to accelerate the identification
process, improve the accuracy of taxonomic assignments,
refine our understanding of which epidermal features
are most appropriate for taxonomic and environmental
reconstructions, and will facilitate investigations of the
functional and ecological significance of different epidermal characters.
We are creating this database of images to facilitate
the development of these potential new methods. We are
48
focusing on the leaf surface, but intend to include cuticle
from all plant parts as the database develops. It will first
focus on fossil material that can be compared directly to
modern relatives, and at a future date we will be able to
expand this to extinct plants (genera and families) that
are also abundant in angiosperm history. While a lack of
comparative modern cuticle material is a problem, large
archives of cuticle preparations are not of practical use
unless the images are sorted into categories. The software
developed for Cuticle allows for the comparison of large
numbers of fossil and modern cuticle specimen images
because they are placed into bin categories defined by
sets of epidermal characters visible on both modern and
fossil material. The database relies on an identification
key that is independent of taxonomy and uses easily
identified characters that have the highest potential for
defining natural groups.
Classification Schemes
The first identification key structure for cuticle was created by Meyen (1966) using a limited four-character-state
system based solely on stomata [stomatal type, stomata
sunken, stomata in bands or dispersed, stomata in sulci
(grooves)], designed specifically for gymnosperms. To create an identification system for angiosperm cuticle that was
independent of initial taxonomic assignment, Roselt &
Schneider (1969) focused on the mature stomatal complex
type, and the distribution of stomata on the leaf surface,
morphological structure of trichomes, and the presence/absence of papillae and glands. Roselt & Schneider (1969)
used multiple states of these stomatal characters to classify
Eocene dispersed cuticle, and then established form genera
based upon characters such as the shape and structure of
trichomes, and presence/absence of papillae and glands.
An artificial classification system is preferable to a
phylogenetic system because it avoids bias from previous taxonomic assignment. This allows the epidermal
characters to define the groups, which can then be compared to recent phylogenies to see how well they match.
We recognized that the expanded character set of Roselt
& Schneider (1969) is more useful than the original four
characters used by Meyen (1966), but also realized that
Roselt & Schneider’s (1969) system is too simplified to
incorporate cuticle from all types of land plants. Thus, we
found it necessary to make significant modifications in
the development of the identification structure of Cuticle
(fig. 4). The identification key structure used in Cuticle is
also available on the web page http://fm1.fieldmuseum.
org/cuticle/PaleoCollaborator under the Project Background tab, and then under the Leaf Description & ID
Tools tab, and will be revised as our understanding of
epidermal characters improves.
Roselt & Schneider (1969) used the stomatal complex type as their first character. Determining the mature
Cour. Forsch.-Inst. Senckenberg, 258, 2007
stomatal type is often difficult for modern material (for
the reasons given in the section on the utility of the
stomatal complex), and is especially difficult for fossil
material despite pristine preservation. As a result we have
placed the stomatal complex type at the end of the key,
with more easily recognized characters first. The identification key structure of Cuticle includes the ability to
classify cuticle from seeds, flowers, stems, and roots (first
node), but the focus is currently on leaf cuticle (point 1
– fig. 4). The second node separates out cuticle that does
not preserve the outlines of epidermal cells needed later
in the key. In most cases this is due to poor preservation
or the lack of cutin deposited along the anticlinal walls
of the epidermal cells (Dilcher 1974), but in some cases
is due to a high density of trichomes on the epidermis
(point 2 – fig. 4). Epidermal characters will eventually
be linked to macrofossil venation patterns, by connecting to the key structure of the Leaf Architecture Working
Group (Ash et al. 1999, point 3 – fig. 4). The presence or
absence of trichomes is used as the fourth node (point 4
– fig. 4) because trichome cells (or their traces) are often
preserved on fossil material, (macrofossil compressions
and dispersed cuticle), and this character is often reported
in the paleobotanical literature. Intact trichome cells are
less commonly preserved on dispersed cuticle because of
abrasion during transport, but even if the trichome cells
are lost, they leave behind a ‘scar’ or trichome base, often
with a characteristic pattern of surrounding epidermal
cells that can be of taxonomic use (Dilcher 1961, Fahn
1982, Upchurch 1995). The fifth node focuses on the
presence or absence of stomata (point 5 – fig. 4). Most
modern plant species are hypostomatous, having stomata
only on the abaxial surface (Metcalfe & Chalk 1950,
Fahn 1982). If the cuticle preserves both leaf surfaces
and the cuticle is amphistomatous, this can provide further diagnostic information regarding the specimen’s
taxonomic affinity (Metcalfe & Chalk 1950). It is noteworthy that stomatal distribution on the leaf surfaces can
vary with environmental conditions. For instance, some
plant species that are normally hypostomatous at low
to moderate elevations can be amphistomatous at high
elevation (Woodward 1986).
Stomatal arrangement is the sixth node (point 6 – fig.
4). Many gymnosperms, in particular conifers, tend to
organize their stomata in rows, but this is not always the
case, nor is this character state restricted to gymnosperms.
Certain species of monocots also have stomata that are
aligned in rows (Wilde et al. 2005, Kvaček & Herman
2004). Stomatal rows can also be grouped together in
multiple rows, known as bands, forming the seventh
node (point 7 – fig. 4). Stomata can be located below the
epidermal pavement, raised or sunken into pits or cavities
(Fahn 1982, Upchurch 1995) (eighth node, fig. 4). These
pits can be covered by dense growth of trichomes as in
Xanthorrhoea (grass tree), and have been interpreted as
an adaptation to arid or semi-arid conditions (Fahn 1982).
Papillate cells are easy to distinguish on both modern and
eschweizerbartxxx sng-
fossil material, and are thus used as the ninth node of the
key (point 9 – fig. 4). Their presence can indicate low
light intensity in an understory habit (Lee 1986, Feild
et al. 2004), or high water stress in a saline or droughtstricken environment (Axsmith & Jacobs 2005, Lydon et
al. 2003, Watson & Alvin 1999). However, certain types
of papillae are strongly associated with a limited set of
taxonomic groups and thus have greater taxonomic value
than paleoecological use (such as papillae which overarch
the stomata in the Sapindaceae). The first nine epidermal
characters utilized in the identification key of Cuticle
are readily identified, easy to classify, are reported in the
botanical literature, and have a high potential for taxonomic and environmental significance. Because of the
difficulties inherent in classification, the final epidermal
character used in Cuticle is the mature stomatal complex
type (node 10, fig. 4), using the fourteen morphological
stomatal complex types recognized by Baranova (1987,
1992) (figs 2 and 3).
PaleoCollaborator Software
The computer software used to organize the images in
Cuticle, called PaleoCollaborator, was designed specifically for comparison of large databases of images and the
associated metadata. It was originally designed for the
comparison of megafossils from the Green River Formation using characteristics of leaf venation, and was engineered to be adaptable to all types of modern and fossil
plant material.
The identification key structure from the Green River
Paleobotany Project (http://greenriver.dmns.org) was
modified to accommodate the different set of characters
used when comparing cuticle specimens. The identification key in Cuticle uses dichotomous (or sometimes
polychotomous) character states to define a set of bin
categories, where species with similar epidermal characters are placed together (fig. 4). The identification key can
be accessed through the Identification Tab that is visible
at the top of every web page. The user navigates through
the identification key on successive web pages, reaching
the next step in the key by choosing between the options
of the character states. Each character state is illustrated
with a black and white line drawing and a photograph
of a modern species from the database. At any point in
the key, a user can view all cuticle specimens that fit the
criteria at that level by selecting a ‘Show All’ link next
to the character (see fig. 1B). For example, if the user
wants to see all cuticular specimens in the database with
cellular preservation, then they would choose: ‘Plant
Cuticle’, then ‘Leaf Cuticle’, and then ‘Show All’ (fig.
1C) next to the ‘Cells Distinct’ link. The resulting output
would show thumbnails of all cuticle images with cellular
preservation stored in the database below point 3 on fig.
4, subdivided into the final bin categories.
49
Barclay et al.: The Cuticle Database: Developing an interactive tool for study of the fossil cuticle record
eschweizerbartxxx sng-
50
Cour. Forsch.-Inst. Senckenberg, 258, 2007
Alternatively, a user can follow the identification key
to the end of one of the branches (fig. 1B). As an example,
if the user wanted to find other cuticle specimens that have
similar characteristics to Ocotea cudadontissi (FLMNH
#253) (fig. 1D), they would need to choose the character
states that lead from choice 1 to choice 6 and then from
7A to 10A on the flow chart in fig. 4. Selections leading
to Ocotea cudadontissi are leaf cuticle, cellular preservation with trichome bases present, stomata dispersed on
the lamina and not sunken into pits, lacking papillae,
and having a paracytic stomatal complex. The resulting output would show thumbnails of all cuticle images
with these characteristics. Selecting one of the thumbnail
images takes the user to the Detail Image page (fig. 1D)
that shows an image of the cuticle (both upper and lower
surfaces when available) as well as associated information such as species name, collector and date, and cuticle
source. Finally, selecting the image on the Detail Page
displays a high-resolution image that can be freely downloaded from the site. While the identification key is the
backbone of Cuticle, all uploaded fossil and modern cuticular images can be searched using criteria on the ‘Quick
Search’ tab (e.g. plant family or specimen number).
Material for Cuticle
A major source of material for the images in Cuticle
is the collection of several thousand prepared slides of
modern cuticle produced by D. Dilcher and his research
laboratory over a 40–year period. These slides and the
leaves from which they were prepared are stored in the
Paleobotany and Palynology collections of the Florida
Museum of Natural History in Gainesville, Florida. This
collection includes vouchered leaf specimens sampled
with permission from herbarium collections, primarily
from The Field Museum, the Munich Herbarium (Botanische Staatssammlung München), and the United States
National Museum, but also from the Fairchild Tropical
Botanic Garden, Kew Gardens, the University of Illinois
at Urbana-Champaign, the University of Indiana, the
Missouri Botanical Garden, and Yale University. The
entire modern reference cuticle collection contains 6146
modern leaf specimens, representing 4208 species in
1680 genera and 190 plant families. Of these, approximately 33% of the vouchered leaf specimens have slides
prepared of their cuticle, which includes 66% of the
represented families. This modern leaf reference collec-
tion was established to study the epidermal and venation
features of extant angiosperms in a systematic fashion,
for comparative work with fossil material, and as a result
some families are well represented while others are represented by only a few taxa.
Digitization of the Florida Museum cuticle collection has occurred in several phases. The pre-existing set
of photographs has been digitally scanned, and we are
currently photographing the existing prepared slides of
abaxial and adaxial leaf cuticle using brightfield microscopy. The adaxial and abaxial images are displayed side
by side on the detail page in Cuticle for comparison (fig.
1D). The ultimate goal of this ongoing database project is
to link microscopic epidermal characters to macroscopic
leaf architecture patterns. As an initial step we are also
photographing the source leaf for the cuticle preparations,
and have future plans to link this cuticle work to the leaf
venation framework created by the Leaf Architecture
Working Group (Ash et al. 1999).
The collection at the Florida Museum is heavily
weighted towards angiosperms. The ten most extensively
sampled families in order of number of species are:
Fagaceae (266), Lauraceae (137), Apocynaceae (121),
Sapindaceae (107), Moraceae (92), Fabaceae (70), Euphorbiaceae (62), Arecaceae (53), Myricaceae (35), and
Araceae (31). In order to improve the taxonomic coverage of Cuticle we will seek additional sources of cuticle
preparations from national and international collections.
eschweizerbartxxx sng-
Conclusions
Attempts to determine which characters are most appropriate for taxonomic identification or conversely inferring
paleoenvironmental conditions have been hindered by
limited access to modern comparative material. Several
large collections of prepared cuticles do exist in paleobotanical collections in North America and Europe, but they
remain largely inaccessible to the research community.
The development of this cuticle database will begin to
centralize these collections, placing images of the material within an identification key structure that allows for
comparison of fossil material with modern material using
the same characteristics. Because Cuticle is available via
the Internet, paleobotanical researchers will have a more
rapid means of comparing their unknown specimens with
images of vouchered herbarium specimens. Being able
to rapidly identify fossil cuticular specimens will also
Fig. 4: Flow chart of Identification Key of Cuticle Database. Successive decisions in the key take the user to a new web page with
instructions and images. The characters used for the identification path to reach the terminus of the key are numbered in circles:
1) Leaf cuticle or other? 2) Cellular detail preserved? 3) Connection to macrofossil venation ID key: one option leads to LAWG
database, the other leads to cuticle characters. 4) Trichomes/trichome bases present? 5) Stomata present? 6) Stomata in rows or
dispersed? 7) Rows single or bands? 8) Stomata sunken in pits? 9) Papillae present? 10) Mature stomatal complex type? Gray boxes
are terminations in the key.
51
Barclay et al.: The Cuticle Database: Developing an interactive tool for study of the fossil cuticle record
benefit researchers concerned with estimating the paleoenvironmental conditions of their fossil assemblages,
potentially leading to the development of novel methods
for understanding how epidermal characters are influenced by environmental parameters.
Acknowledgements
Funding from an EU Marie Curie Excellence Grant
(MEXT-CT-2006-042531) and United States National
Science Foundation grants (0207440 and EAR0643290),
and startup funding from a Packard Fellowship subaward provided by Dr. Peter Wilf of Penn State are
gratefully acknowledged. We would like to thank the IT
department at The Field Museum for hosting Cuticle, and
Peter Lowther specifically for assistance in maintenance
and upgrades. Thomas and Richard Dilhoff from the
Evolving Earth Foundation were integral in funding the
development of the PaleoCollaborator software, designed
by Steve Wagner and Kirk Johnson of the Denver Museum of Nature & Science. Startup funding paid for the
imaging of the modern cuticle image collection by undergraduate students Lenhs Louis and Nakia Wilson at the
University of Florida, whose hard work has made Cuticle
a reality. We thank Terry Lott at the Florida Museum of
Natural History for managing students and for data management and quality control. The effort of undergraduate
student Colin Carney from Northwestern University was
instrumental in processing and loading the images from
Florida into Cuticle. The website home page of Cuticle
was designed by Jessica Leon-Guerrero.
eschweizerbartxxx sng-
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Manuscript submitted 2006–09–08
Manuscript accepted 2007–07–16
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