Sugiol is a phenolic abietane derivative of ferruginol and can be used as a biomarker for specific families of conifers. The presence of sugiol can be used to identify the ''
Cupressaceae
Cupressaceae is a conifer family, the cypress family, with worldwide distribution. The family includes 27–30 genera (17 monotypic), which include the junipers and redwoods, with about 130–140 species in total. They are monoecious, subdio ...
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
s. The polar terpenoids are among the most resistant molecules to degradation besides n-alkanes and
fatty acid
In chemistry, particularly in biochemistry, a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, ...
s, affording them high viability as
biomarker
In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, p ...
s due to their longevity in the sedimentary record. Significant amounts of sugiol has been detected in fossil wood dated to the
Eocene
The Eocene ( ) Epoch is a geological epoch that lasted from about 56 to 33.9 million years ago (mya). It is the second epoch of the Paleogene Period in the modern Cenozoic Era. The name ''Eocene'' comes from the Ancient Greek (''ēṓs'', " ...
and
Miocene
The Miocene ( ) is the first geological epoch of the Neogene Period and extends from about (Ma). The Miocene was named by Scottish geologist Charles Lyell; the name comes from the Greek words (', "less") and (', "new") and means "less recent" ...
periods, as well as a sample of ''Protopodocarpoxylon'' dated to the middle
Jurassic
The Jurassic ( ) is a Geological period, geologic period and System (stratigraphy), stratigraphic system that spanned from the end of the Triassic Period million years ago (Mya) to the beginning of the Cretaceous Period, approximately Mya. The J ...
.
Background
Sugiol is a naturally occurring phenolic diterpenoid. Diterpenoids are a group of secondary metabolites with 20 carbons. Acyclic diterpenes are uncommon, due to the way that they are assembled, and include important molecules such as phytol. Sugiol has three six-membered rings, one of which is aromatic (ring C), and differs from ferruginol only by an addition of an oxo group bound to ring B. It may also be classified as an abietane, a class of tricyclic diterpenoids that share the same basic structure and are commonly found in the resin of
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
s among other terrestrial plants.
Aromatic abietanes that contain an aromatic carbon ring, such as sugiol and ferruginol, have exhibited a variety of interesting properties that have made them of high interest to the pharmacological community. Sugiol specifically has demonstrated anti-tumor, anti-microbial,
antioxidant
Antioxidants are compounds that inhibit oxidation, a chemical reaction that can produce free radicals. This can lead to polymerization and other chain reactions. They are frequently added to industrial products, such as fuels and lubricants ...
, and anti-viral activities.
Sugiol has been shown to inhibit the oncogenic protein STAT3, which is constituently on in malignant tumors. Sugiol directly inhibits the enzyme transketolase, leading to a build up of
reactive oxygen species
In chemistry, reactive oxygen species (ROS) are highly reactive chemicals formed from diatomic oxygen (). Examples of ROS include peroxides, superoxide, hydroxyl radical, singlet oxygen, and alpha-oxygen.
The reduction of molecular oxygen ...
(ROS) and stress-induced cell death. Reactive oxygen species are highly reactive, and can damage cellular mechanisms by oxidizing critical molecules.
Sugiol downregulates inflammatory genes such as NF-κB, COX-2, TNF-alpha, IL-1beta, and IL-6.
Sugiol prevents virus triggered cytopathic effects as a result of H1N1 in MDCK cells for up to 72 hours. It has also been shown to possess significant neutralizing activity against gram-positive and gram-negative bacteria, with slightly higher activity against gram-positive organisms.
Many plant derived compounds have demonstrated potential as therapeutic tools. In one study sugiol showed efficacy in treating '' Leishmania infantum,'' a parasite that can cause Leishmaniasis in humans. Free sugiol was able to induce cell-death in the parasitic bacteria, and when encased in cell walls obtained from yeast was able to enter a parasitized macrophage and inhibit the ''L. infantum'' within.
Because sugiol has shown so many protective effects in therapeutic trials, it is likely that in plants it acts as a chemical defense agent. Sugiol present in the resins of conifers may help to protect the plant against ROS generated during metabolism, as well as against any pathogenic viruses or bacteria.
Reaction pathways
Diterpenes are commonly synthesized from the precursor molecule geranylgeranyl pyrophosphate (GGPP). GGPP's hydrocarbon backbone can be rearranged into different structures that may be further rearranged or added to in order to create precursors for different families of diterpenoid compounds. This precursor molecule may be synthesized through the mevalonic acid pathway or the deoxyxylulose pathway. These pathways produce isopentenyl pyrophosphate, which can be rearranged into GGPP. The cyclization of GGPP and the subsequent reorganizations into different precursors is controlled by a large family of enzymes known as diterpene syntheses (diTPS).
To synthesize sugiol a plant must first synthesize GGPP through either of the previously mentioned pathways, ( mevalonic acid or the deoxyxylulose pathway), then rearrange GGPP into the molecule mitiradiene. After formation of an intermediate compound abietatriene, a
cytochrome P450
Cytochromes P450 (CYPs) are a superfamily of enzymes containing heme as a cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are important for the clearance of various compo ...
enzyme can then attach an oxygen molecule to the intermediate. This produces ferruginol, which can then be modified to sugiol by sugiol synthase.
Sugiol may then be formed through the modification of ferruginol according to the following reaction driven by the enzyme sugiol synthase.
:
Plant sources
Abietanes may fall into one of two classes, either regular or
phenol
Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it ...
ic. Regular abietanes are common across all
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
s, whereas phenolic abietanes are usually found in more specific families and are mostly absent from
pinaceae
The Pinaceae, or pine family, are conifer trees or shrubs, including many of the well-known conifers of commercial importance such as cedars, firs, hemlocks, larches, pines and spruces. The family is included in the order Pinales, formerly know ...
. There are a few exceptions to this, including detection of ferruginol and its derivative in ''
Cedrus atlantica
''Cedrus atlantica'', the Atlas cedar, is a species of tree in the pine family Pinaceae, native to the Rif and Atlas Mountains of Morocco ( Middle Atlas, High Atlas), and to the Tell Atlas in Algeria.Gaussen, H. (1964). Genre ''Cedrus'' ...
Cupressaceae
Cupressaceae is a conifer family, the cypress family, with worldwide distribution. The family includes 27–30 genera (17 monotypic), which include the junipers and redwoods, with about 130–140 species in total. They are monoecious, subdio ...
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
families. It has not been significantly detected in
Pinaceae
The Pinaceae, or pine family, are conifer trees or shrubs, including many of the well-known conifers of commercial importance such as cedars, firs, hemlocks, larches, pines and spruces. The family is included in the order Pinales, formerly know ...
. Similar phenolic abietanes have also been detected in cedars (genus ''
Cedrus
''Cedrus'', common English name cedar, is a genus of coniferous trees in the plant family Pinaceae (subfamily Abietoideae). They are native to the mountains of the western Himalayas and the Mediterranean region, occurring at altitudes of 1,500� ...
''), pines (genus ''
Pinus
A pine is any conifer tree or shrub in the genus ''Pinus'' () of the family Pinaceae. ''Pinus'' is the sole genus in the subfamily Pinoideae. The World Flora Online created by the Royal Botanic Gardens, Kew and Missouri Botanical Garden a ...
''), monkey puzzle (genus '' Araucaria''), and torreya (genus ''
Torreya
''Torreya'' is a genus of conifers comprising six or seven species placed in the family Taxaceae, though sometimes formerly placed in Cephalotaxaceae. Four species are native to eastern Asia; the other two are native to North America. They ...
''). Sugiol has also been detected in certain
angiosperm
Flowering plants are plants that bear flowers and fruits, and form the clade Angiospermae (), commonly called angiosperms. They include all forbs (flowering plants without a woody stem), grasses and grass-like plants, a vast majority of br ...
Melia Melia or Melie ( Ancient Greek: Μελία, Μελίη) may refer to:
Greek mythology
* Melia (mythology), the name of several figures
* Melia, the singular form of Meliae, a type of nymph
People and fictional characters
* Melia (given name)
* ...
'', but is much more prevalent in
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
s. This allows for these organisms to be excluded from the list of species for which sugiol is a
biomarker
In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, p ...
. The enzyme sugiol synthase has also been isolated from '' Salvia militiorrhiza'', an
angiosperm
Flowering plants are plants that bear flowers and fruits, and form the clade Angiospermae (), commonly called angiosperms. They include all forbs (flowering plants without a woody stem), grasses and grass-like plants, a vast majority of br ...
that contains high levels of phenolic diterpenes and is commonly utilized in
traditional Chinese medicine
Traditional Chinese medicine (TCM) is an alternative medicine, alternative medical practice drawn from traditional medicine in China. It has been described as "fraught with pseudoscience", with the majority of its treatments having no logica ...
.
Preservation
Organic compound
In chemistry, organic compounds are generally any chemical compounds that contain carbon- hydrogen or carbon-carbon bonds. Due to carbon's ability to catenate (form chains with other carbon atoms), millions of organic compounds are known. Th ...
s originally in living organisms can be preserved in the rock record if certain requirements are met. Proper preservation requires ample supply of organic material, high burial of that organic matter, and that the organic matter is then polymerized and not degraded. The more degraded a
biomolecule
A biomolecule or biological molecule is a loosely used term for molecules present in organisms that are essential to one or more typically biological processes, such as cell division, morphogenesis, or development. Biomolecules include larg ...
is the less specific of a biomarker it becomes, as multiple molecules may have the same
hydrocarbon
In organic chemistry, a hydrocarbon is an organic compound consisting entirely of hydrogen and carbon. Hydrocarbons are examples of group 14 hydrides. Hydrocarbons are generally colourless and hydrophobic, and their odors are usually weak or ...
skeleton after
diagenesis
Diagenesis () is the process that describes physical and chemical changes in sediments first caused by water-rock interactions, microbial activity, and compaction after their deposition. Increased pressure and temperature only start to play ...
. However, polar terpenoids such as sugiol may be preserved in their unaltered forms in fossil conifers, potentially due to plant resins that protect them from degradation.
In samples obtained from a Pliocene fossilized forest most molecules had been significantly degraded, but phenolic abietanes including sugiol remained intact and identifiable. Even in samples that had been approximately 37.7% decomposed as determined by comparing cellulose content, trace amounts of sugiol and more than 10% ferruginol were detected via GC/MS. Sugiol will remain detectable in a sample long after it has lost its anatomical identifiers, making it extremely useful in identifying extremely old or decomposed plant fossils.
In a study of preserved fossil wood and buried samples from a middle
Jurassic
The Jurassic ( ) is a Geological period, geologic period and System (stratigraphy), stratigraphic system that spanned from the end of the Triassic Period million years ago (Mya) to the beginning of the Cretaceous Period, approximately Mya. The J ...
forest located in Poland, a negative correlation was observed between the preservation of anatomical features of the plant samples versus the chemical features. It was hypothesized that the rapid
mineralization
Mineralization may refer to:
* Mineralization (biology), when an inorganic substance precipitates in an organic matrix
** Biomineralization, a form of mineralization
** Mineralization of bone, an example of mineralization
** Mineralized tissues are ...
processes required to preserve biomolecules degraded the organic matter, but either extracted or trapped chemical biomarkers in the clay mineral matrix during the early stages of
mineralization
Mineralization may refer to:
* Mineralization (biology), when an inorganic substance precipitates in an organic matrix
** Biomineralization, a form of mineralization
** Mineralization of bone, an example of mineralization
** Mineralized tissues are ...
, protecting those molecules from breakdown. Burial of samples in
anaerobic
Anaerobic means "living, active, occurring, or existing in the absence of free oxygen", as opposed to aerobic which means "living, active, or occurring only in the presence of oxygen." Anaerobic may also refer to:
*Adhesive#Anaerobic, Anaerobic ad ...
sediments decreased biodegradation and increased preservation of biomarkers including sugiol. Sugiol was significantly more abundant in less oxidized samples. Additionally, the antimicrobial properties of sugiol could help to decelerate biodegradation of itself and other natural products by decreasing microbe driven breakdown.
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is u ...
(MS) are commonly used to detect and identify sugiol in a sample. GC/MS is highly specific and sensitive and allows for identification of a wide range of analytes. After extraction from the original sample, which could be the
resin
In polymer chemistry and materials science, resin is a solid or highly viscous substance of plant or synthetic origin that is typically convertible into polymers. Resins are usually mixtures of organic compounds. This article focuses on n ...
of a living plant, or a preserved rock sample, the sample can be ionized and the components identified through their representative spectra. Analysis of fragmentation patterns can also be used to identify a compound by connecting each peak in the mass spectra to the masses of significant
fragmentation
Fragmentation or fragmented may refer to:
Computers
* Fragmentation (computing), a phenomenon of computer storage
* File system fragmentation, the tendency of a file system to lay out the contents of files non-continuously
* Fragmented distributi ...
products of the molecule, as well as the molecular ion, which is the largest significant peak in the spectra.
When identifying sugiol in a sample, full-scan monitoring is commonly used to scan the full range of masses from 50 to 650 Da. This allows for detection of compounds with a wide range of molecular masses when attempting to make an identification based on chemical composition.
Electron impact ionization
Electron ionization (EI, formerly known as electron impact ionization and electron bombardment ionization) is an ionization method in which energetic electrons interact with solid or gas phase atoms or molecules to produce ions. EI was one of th ...
is also commonly used to break apart and ionize the samples before they are passed to the mass spectrometer.
The molecular ion peak for sugiol appears as a small peak at an m/z ratio of 300.2084. The largest peak in the mass spectra appears at a m/z ratio of 285.1849, and corresponds to a fragmentation product with a formula of C19H25O2. This fragmentation product has one less ring and an H2O molecule bound to the newly open carbon chain. Another significant peak is at m/z 257.1536, and corresponds to another
fragmentation
Fragmentation or fragmented may refer to:
Computers
* Fragmentation (computing), a phenomenon of computer storage
* File system fragmentation, the tendency of a file system to lay out the contents of files non-continuously
* Fragmented distributi ...
product with a single ring, and a formula of C17H21O2.{{Cite web, title=SUGIOLE - MS - Spectrum - SpectraBase, url=https://spectrabase.com/spectrum/Dr4tLse9t1E, access-date=2021-05-20, website=spectrabase.com Further significant peaks appear at m/z's of 217 and 243, corresponding to formulas of C13H15O2 and C15H19O2 respectively.
Derivitization
Sugiol is a protic molecule. Protic molecules are those that have protic groups or hydrogen molecules that readily leave the molecule, such as -OH, -NH, and -HF. These molecules can complicate GC/MS data by increasing peak tailing and affecting the ease with which they can be separated by the GC. In order to avoid this effect, protic molecules are often subjected to derivatization reactions, in which the offending protons are replaced by a different
functional group
In organic chemistry, a functional group is a substituent or moiety in a molecule that causes the molecule's characteristic chemical reactions. The same functional group will undergo the same or similar chemical reactions regardless of the res ...
. A commonly used replacement group is trimethylsilyl (TMS), which produces trimethylsilyl derivatives of the original protic molecules. Another commonly used group is ''tert''-butyldimethylsilyl (TBDMS), also used to derivatize hydroxyl and amine protic groups. Diazomethane has also been used to form methyl esters from carboxylic acids.
Case studies
The combination of the longevity of sugiol in environmental samples and its presence in only specific families of plants make it an excellent biomarker. Detection of sugiol in combination with other biomarkers like ferruginol or other diterpenes can also help to bolster the identification of the sample, as well as to narrow the scope of possible identities to only a few specific
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
families. Sugiol has been utilized in the identification of extinct plant taxa such as ''Protopodocarpoxylon'', and ''Taxodioxylori gypsaceum''.
Identification of ''Protopodocarpoxylon''
''Protopodocarpoxylon'' is an extinct
genus
Genus ( plural genera ) is a taxonomic rank used in the biological classification of living and fossil organisms as well as viruses. In the hierarchy of biological classification, genus comes above species and below family. In binomial n ...
of
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
tracheophytes, now often found as fossilized woods. In a 2007 study, extraction and identification of
biomarker
In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, p ...
s from fossil woods collected in south-central
Poland
Poland, officially the Republic of Poland, , is a country in Central Europe. Poland is divided into Voivodeships of Poland, sixteen voivodeships and is the fifth most populous member state of the European Union (EU), with over 38 mill ...
allowed for the identification of the sample as ''Protopodocarpoxylon'' Eckhold''.'' Samples of the wood were collected from clays and carbonate concretions then cleaned of contaminants before being pulverized, and the organics extracted. The extracts were derivatized with TMS and then subjected to gas chromatography-mass spectrometry (GC-MS) analysis.
Multiple abietanes were detected in the analyzed samples, with ferruginol, sugiol, simonellite, and dehydroabietane present in all four of the samples tested. Sugiol and ferruginol were both detected as unaltered natural products. There was a dramatic difference in detected abundance of sugiol and ferruginol in samples that were more oxidized, but the biomarkers were still detectable in both cases.
The unknown fossil wood samples were determined to contain aliphatic lipids (''n''-alkanols and ''n''-alkanoic acids), diterpenoids (abietanes, labdanes, and totaranes), triterpenoids (lupane and hopane), and
steroid
A steroid is a biologically active organic compound with four rings arranged in a specific molecular configuration. Steroids have two principal biological functions: as important components of cell membranes that alter membrane fluidity; and ...
s. The presence of long chain ''n''-alkanes, ferruginol, sugiol, and dehydroabietic acid were considered and the sample was determined to be a
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
Cupressaceae
Cupressaceae is a conifer family, the cypress family, with worldwide distribution. The family includes 27–30 genera (17 monotypic), which include the junipers and redwoods, with about 130–140 species in total. They are monoecious, subdio ...
'', or ''
Araucariaceae
Araucariaceae – also known as araucarians – is an extremely ancient family of coniferous trees. The family achieved its maximum diversity during the Jurassic and Cretaceous periods and the early Cenozoic, when it was distributed almost world ...
'' family. All of these chemical identifiers, combined with distinct morphological features characteristic of tracheids allowed for the assignment of ''Protopodocarpoxylon'' to the sample.
The presence of multiple biomarkers, each of which correspond to different groups of organisms allows potential identities to be narrowed down. When combined with phenotypic characteristics, specific biomarkers like sugiol become very strong tools in identifying unknown organisms.
Identification of ''Taxodioxylori gypsaceum''
''Taxodioxylon gysaceum'' is an extinct species of
conifer
Conifers are a group of cone-bearing seed plants, a subset of gymnosperms. Scientifically, they make up the division Pinophyta (), also known as Coniferophyta () or Coniferae. The division contains a single extant class, Pinopsida. All ex ...
presently found as fossil wood.
Samples of the same wood in various stages of degradation were collected from a forest in Italy, originally existing during the
Pliocene
The Pliocene ( ; also Pleiocene) is the epoch in the geologic time scale that extends from 5.333 million to 2.58 These samples were milled and filtered into different fractions by coarseness before steam distillation was utilized to extract
terpene
Terpenes () are a class of natural products consisting of compounds with the formula (C5H8)n for n > 1. Comprising more than 30,000 compounds, these unsaturated hydrocarbons are produced predominantly by plants, particularly conifers. Terpenes ar ...
s. The extraction was then analyzed through GC/MS. The comparative degree of degradation was determined by analysis of holocellulose contents in each sample. Holocellulose refers to the fraction of plant biomass that includes cellulose and hemicellulose but excludes lignin. These carbohydrates are broken down during decomposition, and so their concentrations can be used as a measure of the degree of degradation.
A variety of
terpene
Terpenes () are a class of natural products consisting of compounds with the formula (C5H8)n for n > 1. Comprising more than 30,000 compounds, these unsaturated hydrocarbons are produced predominantly by plants, particularly conifers. Terpenes ar ...
s were detected in the degraded lignite samples, including more than 10% ferruginol, between 5 and 10% podocarpodiol, and less than 5% of sugiol. These compounds were hypothesized to have become more prevalent in the degraded sample due to preferential decomposition of other compounds. The presence of these
terpene
Terpenes () are a class of natural products consisting of compounds with the formula (C5H8)n for n > 1. Comprising more than 30,000 compounds, these unsaturated hydrocarbons are produced predominantly by plants, particularly conifers. Terpenes ar ...
s in this sample suggest that the organism belongs to the ''
Cupressaceae
Cupressaceae is a conifer family, the cypress family, with worldwide distribution. The family includes 27–30 genera (17 monotypic), which include the junipers and redwoods, with about 130–140 species in total. They are monoecious, subdio ...
terpene
Terpenes () are a class of natural products consisting of compounds with the formula (C5H8)n for n > 1. Comprising more than 30,000 compounds, these unsaturated hydrocarbons are produced predominantly by plants, particularly conifers. Terpenes ar ...
s present, the sample was identified as ''Taxodioxylon gypsaceum''''.'' This combination of
terpene
Terpenes () are a class of natural products consisting of compounds with the formula (C5H8)n for n > 1. Comprising more than 30,000 compounds, these unsaturated hydrocarbons are produced predominantly by plants, particularly conifers. Terpenes ar ...
s has also been detected in other samples known to be ''Taxodioxylon gypsaceum'', further supporting this identification.
The yields of
terpene
Terpenes () are a class of natural products consisting of compounds with the formula (C5H8)n for n > 1. Comprising more than 30,000 compounds, these unsaturated hydrocarbons are produced predominantly by plants, particularly conifers. Terpenes ar ...
s retrieved from these samples were higher than other species that also contain
phenol
Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it ...
ic diterpenes, suggesting that high percentages of
sesquiterpene
Sesquiterpenes are a class of terpenes that consist of three isoprene units and often have the molecular formula C15H24. Like monoterpenes, sesquiterpenes may be cyclic or contain rings, including many unique combinations. Biochemical modifica ...
s and diterpenes are an additional biomarker for ''Taxodioxylon gypsaceum''''.''