Plant remains from the Early Cretaceous Fossil-Lagerstätte of Pietraroja, Southern Italy, Benevento

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Influence of volcanic gases on the epidermis of Pinus halepensis Mill. in Campi Flegrei, Southern Italy: A possible tool detecting volcanism in present and past floras

Over the geological history of the planet, among chronic environmental stress factors advocated as killing agents (Visscher et al., 2004), changes in atmospheric chemistry had world-wide dramatic effects on plant life in land (e.g. Visscher et al., 1996; Meyer and Kump, 2008). For instance, among chemical contaminants that could have disrupted end-Permian biota, volcanogenic SO2 (Visscher et al., 2004) and biological H2S (euxinia mechanism: Kump et al., 2005; Berner and Ward, 2006) gases are favoured to explain extinction. In particular, volcanism subsequently played a role in both maintaining and perturbing the atmosphere chemistry and physics, with important implications in terms of the evolution of life (Mather, 2008). The development of large igneous province (LIP) and continental flood basalt province (CFBP) (Courtillot and Renne, 2003; Jerram et al., 2005; Keller, 2008; Bryan et al., 2010) commonly coincides with mass extinction events (Wignall, 2001, 2005; Rampino, 2010; Whiteside et al., 2010) and results in the release of significant volumes of gases, such as CO2, H2S and SO2 into the atmosphere (Beerling and Berner, 2002; Berner and Beerling, 2007; Hori et al., 2007). It is widely recognized that volcanic sulfur dioxide (SO2) and hydrogen sulfide (H2S) emissions are significant sources of sulfur release to the atmosphere (Bates et al., 1992; Berner and Berner, 1996).
Gases emitted by volcanoes represent both a factor inhibiting vegetation development (Whittaker et al., 1989) and could have been responsible of the decline of vegetation during periods of global-scale volcanism (Bond et al., 2010; Visscher et al., 2004; Whiteside et al., 2010; McElwain and Punyasena, 2007). In particular, H2S is often thought to be a phytotoxin, being harmful to the growth and development of plants (Lisjak et al., 2010) especially when the quantities are higher than plant necessity (Thompson and Kats, 1978; Lorenzini and Nali, 2005). Moreover, atmospheric pollutants produced by volcanic activity and OAEs, such as SO2 and H2S, are said to be absorbed via the cuticle as well as the stomata (Haworth and McElwain, 2008).
Plants exposed to poisonous volcanic gases may show signs of diseases to total defoliation and death (e.g. Dickson, 1965; Clarkson and Clarkson, 1994; Delmelle et al., 2002). However, plant damages are related to both gas concentration (Delmelle, 2003) and its persistence (Grattan et al., 1998) in the atmosphere. Under severe pollution conditions, the direct phytotoxic effects of gaseous pollutants as well as long-term effects of acid washout (Grattan and Pyatt, 1994) can even be considered as potential environmental mutagens disturbing plant growth and community structure (Visscher et al., 1996). As a matter of fact, as Visscher et al. (2004) pointed out, variation in structure and composition of leaf cuticles is a potential source of botanical evidence on mutational effects of environmental stress factors.
Therefore, leaves in natural environments are subjected to a range of physical processes which may damage their surfaces, leading to alterations in the structure and integrity of the cuticle, and consequently changes in the physical properties of the leaf surfaces (van Gardingen et al., 1991).
To this end, numerous articles have been published in relation to the effects and interactions of the volcanic activity products (e.g. tephra or ash fall) on both fossil (e.g. Kovar-Eder et al., 2001; García Massini and Jacobs, 2011) and extant plants (Winner and Mooney, 1980b; Cook et al., 1981; Seymour et al., 1983; Dale et al., 2005). Moreover, in extant plants the concentration of chemical elements in the leaves (Notcutt and Davies, 1989; Martin et al., 2009a and b) and the analysis of the log (Baillie and Munro, 1988; Battipaglia et al., 2007) together with field studies led to significant advances in understanding the composition and dispersion of volcanic emissions at source (e.g. Kempter et al., 1996; Delmelle et al., 2002), including major “gas species” (Costa et al., 2005; Chiodini, 2008; Chiodini et al., 2010a).
Leaves of plants act as passive and active collectors for natural (e.g. Martin et al., 2009a) and anthropogenic (e.g. Bačić et al., 1999) airborne pollutants (e.g. gas, aerosols and dusts) and are more sensitive to air quality than other plant organs (e.g. roots) (Landolt et al., 1989; Casseles, 1998; Kabata-Pendias, 2001); the gas exchanges between the plant and the surrounding atmosphere are mediated by the cuticle; this non-living (Riederer, 2006) thin (<0.1-10 m thick in extant plants) and heterogeneous membrane (van Gardingen et al., 1991) covers the epidermis of the aerial part of many tracheophytes (Guignard et al., 2004) and consists of a polymer matrix (cutin), polysaccharides and associated solvent-soluble lipids which are synthesised by the epidermal cells and deposited on their outer wall (Kirkwood, 1999; Riederer and Schreiber, 2001). The outer surface of the cuticle is coated with epicuticular waxes, a general term (Jeffree, 2006) designating very long chain hydrocarbons found embedded within the cuticle and also in the crystalline epicuticular wax layer (Bird and Gray, 2003). The main function ascribed to waxes is to limit the diffusional flow of water and solutes across the cuticle (Heredia and Dominguez, 2009), providing protection for the leaf cells (Turunen and Huttunen, 1990) and acting as the main barrier to air pollutants (e.g. Jeffree, 1986). The composition and amount of waxes in the cuticle have been shown to vary depending to environmental conditions of the plant (Baker, 1982; Bird and Gray, 2003) and according to many authors air pollution seems to increase the rate of wax tubules degradation (e.g. Huttunen and Laine, 1983; Riding and Percy, 1985; Berg, 1987; Turunen and Huttunen, 1990, 1991, Huttunen, 1994). In particular, wax load and structure can be used as an indicator of pollution level (Hansell and Oppenheimer, 2006; Holroyd et al., 2002). The epicuticular wax of pine needles undergoes an ageing procedure during the needle lifetime (Turunen and Huttunen, 1996; Bačić et al., 1999) and is disturbed by polluted air (Huttunen, 1994).
The literature is replete with references to structural changes in epicuticular waxes following exposure to air pollutants (see Turunen and Huttunen, 1990), and as a matter of fact, the erosion of epicuticular waxes is a relevant factor of the multiple forest decline syndrome (Turunen and Huttunen, 1990).
Few paleobotanical works have been achieved on cuticular characters related to volcanic stress. Archangelsky et al. (1995) and Villar de Seoane (2001) studied Early Cretaceous plants from Patagonia (recovered in Baqueró and Springhill Format ions, respectively) demonstrating that the volcanic ash fall played an important role in the formation of xeromorphic structures. As Haworth and McElwain (2008) claimed, the effect of toxic atmospheric gases and volcanic dust would explain xeromorphic features of Pseudofrenelopsis parceramosa (Fontaine) Watson from the Early Cretaceous of England. Moreover, the relationship between ultrastructural characteristics of cuticle and the environment is still poorly understood for extinct as well as extant plants (Guignard et al., 2001) and cuticular ultrastructure data are not numerous for fossil conifers (e.g. Guignard et al., 1998; Villar de Seoane, 1998; Yang et al., 2009) and seem to be still lacking for some species belonging to the genus Pinus (Jeffree, 2006).
However, to date, no studies have been carried out relatively to the response of the ultrastructural features of plant cuticle exposed to the persistent volcanic gases. Conifers are well suited for studies of pollutant levels because they are evergreen and often have long-lived foliage. Usually the needles have a life cycle of several years (Hellström, 2003).
Therefore, the protective role of the epicuticular waxes is particularly important for conifers that have to ensure their investment in leaf tissue for several years (Chabot and Chabot, 1977). In the volcanic area of Pisciarelli (Campi Flegrei, Southern Italy) the gymnosperm Pinus halepensis Mill. (Aleppo pine) is the only conifer growing adjacent to the fumaroles, and much of the surrounding vegetation (under study) displays indications of damage caused by toxic gases. P. halepensis is the most abundant pine species in the western Mediterranean Basin, where it occupies 2.5 million ha (Quézel, 2000) and it is considered as an opportunistic species (Nathan and Ne‟eman, 2000) which is able to regenerate either in the absence or as a result of fire. In addition, P. halepensis has an elevated resistance to drought (Boddi et al., 2002), so much so that Emberger (1930) identifies it as being semiarid, and Oppenheimer (1968) considers it as the most arid-tolerant of all the Pinus species. As a matter of fact, the present study aims to assess the cuticular response of this conifer at a prolonged exposition to the volcanic gases using both SEM and TEM approaches. Moreover, to our knowledge, this is the first study that tests the cuticle ultrastructure behaviour during two subsequent years (current- and first-year-old needles) in response to the fumigation of volcanic gases containing H2S.
In particular, this research aimed to investigate: 1) response of plants to volcanic gases through different aspects: epicuticular and epistomatal waxes and ultrastructural features of the cuticle; 2) potential implications of the conifer cuticle response across environmental stress periods during the geological past; 3) a new method detecting the influence of volcanism for extant and fossil plants.

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Material and methods

The material was collected from two localities in the Phlegrean Fields (Campi Flegrei, Campania Region), an active caldera which spans the last 50000 years (Scandone et al., 2010), characterized by significant recent ground deformation (Morhange et al., 2006) and considered as one of the most dangerous volcanic areas in the world (Chiodini et al., 2010a). In particular, pine needles were recovered from the famous fumaroles field in Pisciarelli locality (40°49‟48.88‟‟N, 14°08‟46.95‟‟E) about 1 km SE of the Solfatara volcano, both characterised by volcanic gas emissions (Fig. 1A,B). Control sample of needles were collected from a volcanic quiescent area (Cigliano: 40°50‟46.46‟‟N, 14°07‟36.31‟‟E) about 2.5 km from Pisciarelli and characterised by the absence of volcanic gas emissions and the presence of clean air. Both localities are characterised by the same soil features (Di Gennaro and Terribile, 1999; Di Gennaro, 2002) and sun exposition and are far away from traffic and industries.
Fig. 1. (A) Location of the Pisciarelli area in the Campania Region. (B) Sketch map showing the location of Solfatara crater and Pisciarelli localities. (C) Close up view of Pisciarelli area with the main fumarole. Dotted line indicates the area of diffuse degassing.
The temperature reaches about 97°C at the Pisciarelli fumaroles (Chi odini et al., 2010a) and the analysis of gaseous compositions (Caliro et al., 2007) revealed that the main component of the fumaroles is H2O followed by CO2, H2S, N2, H2, CH4, He, Ar, and CO. The absence of acidic gases (SO2, HCl, and HF) can be also noted (Chiodini et al., 2010a). Natural high atmospheric concentration of sulphur gas may occur locally in areas with volcanic and geothermic activity (De Kok et al., 2007). As a matter of fact, at Pisciarelli volcanic vent, the high H2S air concentration (equal to ca 600 m/mol) at source (Chiodini et al., 2010a) is also testified by both typical smell (the odour threshold is >0.02 m l-1: De Kok et al., 2007) of rotten eggs in the surrounding air and by indirect corrosive action of this gas -well visible on the iron objects- which caused people to abandon some buildings in the area (Fig. 1C). Nevertheless, this extreme environment is inhabited by very few angiosperm species providing xeromorphic features (e.g. Erica arborea) and the boiling water of fumaroles retains the cyanidialean alga Galdieria phlegrea (Pinto et al., 2007).
Distal volcanic impacts have shown that plants are generally less sensitive to eruptions outside the growing season (Zobel and Antos, 1997; Hotes et al., 2004) and, as Payne and Blackford (2008) pointed out, in winter, plants are senescent and higher rainfall may serve to remove rapidly volcanic pollutants. In case of Pinus halepensis, an evergreen plant permanently fumigated by volcanic gases, retaining leaves for over a year, these “ground noises” do not exist.
The trees present diffuse damages along the North sides of the crown, while needles show symptoms consisting in leaf-tip non-specific discoloration which gradually increasing shootward (terminology from Baskin et al., 2010). Current- and first-year-old needles were collected from branches at heights over 1.5 m from three trees (15-20 years old) at each site (Pisciarelli and Cigliano). Needles were carefully handled to avoid damaging the epicuticular waxes. Following Reed‟s remarks (1982) and also Crang and Klomparens‟ ones (1988) about possible changes in epicuticular wax structures occurring during sample preparation, in order to limit any chemical or physical damages, especially for preserving and dehydrating samples for wax morphology studies (e.g. Turunen and Huttunen, 1991; Tuomisto and Neuvonen, 1993), needles were air-dried for 1-week at mild room temperatures. Among several hundreds of pine needles collected at each site (fumigated and not fumigated), 60 were selected for scanning electron microscope (SEM), then 16 were carefully selected for transmission electron microscope (TEM). Stomata were observed on 15 current- and first-year-old needles for each site. Analysis was performed within two weeks from sampling. Taxonomical identification of García Álvarez et al.
(2009) approach has been used. Light microscope observations were made using a Leitz microscope.
In order to quantify the quality of epicuticular and epistomatal waxes, SEM observations were carried out. Untreated needle sections of approximately 5 mm in length – obtained from the middle of each needle- were mounted on stubs using double-sided adhesive tape; both abaxial and adaxial surfaces have been studied. Part of specimens were sputter-coated with gold using an AGAR Auto Sputter Coater, while the specimens for energy diffractive x-ray (EDX) analyses were coated with carbon in a Emitech K450, observed and photographed with JEOL JSM-5310 SEM adapted with an Energy Diffractive X-ray Oxford Inca X-act at the CISAG (Centro Interdipartimentale di servizi per analisi Geomineralogiche) in the Dipartimento di Scienze della Terra, Università degli Studi di Napoli “Federico II”. The operative conditions were as follows: 25-30 KV accelerating voltage, 100 A emission current, 15 m spot size, 20 mm microscope work distance and 1 min spectra collection time. To quantify the wax change in stomatal chamber, the Nicolotti et al. (2005) needle damage classes have been used as a criterion for the level of crystalline wax degradation.
Samples for TEM were dropped in paraformaldehyde solution mixed in a phosphate-sodium buffer for 3 weeks using Lugardon’s technique (1971), washed and postfixed in a 1% osmium tetroxide solution mixed in a phosphate–sodium buffer for 24 hours. Dehydrated in graded ethanol series during 48 h, the samples were dropped in propylene oxide with an increasing percentage of Epon resin for 24 h. Transferred into pure Epon resin during 24 hours, they were embedded in fresh Epon resin using flat moulds. The preparations were subsequently treated for polymerization at 56 °C for 3 days. Ult rathin (60-70 nm) sections were sectioned with a diamond knife, using a Reichert Ultracut microtome. Ultrathin sections were placed on uncoated 300 Mesh copper grids and stained manually both with a methanol solution of 7% uranyl acetate for 15 min and an aqueous lead-citrate solution for 20 min, then observed and photographed with a Philips CM 120 TEM at 80 kV, in the Centre de technologie des microstructures (CT ) of Lyon-1 University, Villeurbanne, France. Totally 16 pieces of material were embedded in Epon resin blocks. 90 uncoated mesh copper grids were prepared (80 as transversal sections, i.e. perpendicular to the leaf length; 10 as longitudinal sections, i.e. parallel to the leaf length). In order to detect the presence of sulphur in the cuticle, 40 measurements (i.e. 10 measures for each current- and first-year-old needles, fumigated and not fumigated by volcanic gases) with EDS microanalysis were carried out on different parts of the cuticle and also on the cytoplasm remnants of the epidermal cells. The sulphur analysis was performed on 25 coated 300 Mesh grids with transmission electron microscope JEOL 1200EX coupled to a microanalysis system EDS Si(Li) 30mm² NORAN VOYAGER III with an acceleration voltage = 80 kV and a spectres acquisition time = 60 s.
All the quantitative TEM measurements were made with tools in the ImageJ program (Abramoff et al. 2004). The terminology of Holloway (1982) and Archangelsky (1991) was used for the ultrastructural analysis. All specimens and SEM stubs are housed in the Dipartimento di Scienze della Terra, Largo San Marcellino, 10, Napoli, Italy. The resin blocks and TEM negatives are stored in the Lyon-1 University, Villeurbanne, France.

Table of contents :

CHAP. I Influence of volcanic gases on the epidermis of Pinus halepensis Mill. in Campi Flegrei, Southern Italy: A possible tool detecting volcanism in present and past floras
1.1. Introduction
1.2. Material and methods
1.3. Results
1.3.1. Sulphur measures
1.3.2. Scanning electron microscopy observations
1.3.3. Transmission electron microscopy observations
1.4. Discussion
1.4.1. Environmental response of Pinus halepensis to volcanism
1.4.1.1. Epicuticular and epistomatal wax
1.4.1.2. Cuticular membrane (CM) + cell wall (CW)
1.4.2. Potential application for extant and fossil material
CHAP. II The cuticle micromorphology of in situ Erica arborea L. exposed to long-term volcanic gases in Phlegrean Fields, Campania, Italy
2.1. Introduction
2.2. Material and methods
2.2.1. Plant material and sites description
2.2.2. Gas vent
2.2.3. SEM, TEM and EDS preparations
2.2.4. Gas concentration measurements in air and soil
2.2.5. Statistical analysis
2.3. Results
2.3.1. Energy diffractive X-ray analysis with SEM
2.3.2. Scanning electron microscopy observations
2.3.3. Trasmission electron microscope observations
2.4. Discussion
2.4.1. Chemical and SEM considerations
2.4.2. TEM considerations
CHAP. III An Early Cretaceous flora from Cusano Mutri, Benevento, southern Italy
3.1. Introduction
3.2. Geological setting
3.2.1. Stratigraphy
3.3. Material and methods
3.4. Systematic palaeontology
3.5. Taphonomic and palaeoecological remarks
3.5.1. Taphonomy
3.5.2. Palaeoecology of the Cusano Mutri sedimentary basin
3.5.3. Xeromorphic adaptations of the plants
3.5.4. Palaeoclimate and floral comparison
CHAP. IV Plant remains from the Early Cretaceous Fossil-Lagerstätte of Pietraroja, Southern Italy, Benevento
4.4.1. Introduction
4.2. Geological setting
4.3. Material and methods
4.4. Systematic Palaeontology
4.5. Taphonomic and palaeoecological implications
4.5.1. Taphonomy
4.5.2. Palaeoecology of the sedimentary basin
4.5.3. Palaeoclimate and comparison with other Albian florae
General Conclusions

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